At least six international studies have reported T cell reactivity against SARS-CoV-2 in 20% to 50% of people with no known exposure to the virus. Experts suggest doing three tests simultaneously: RTPCR, antibody test, and a T-cell assay, which will give a picture to policymakers if the State or the country has achieved herd immunity against SARS-CoV-2.

A type of white blood cell, T cells are part of the immune system and develop from stem cells in the bone marrow. They help protect the body from infection. Also called T lymphocyte and thymocyte.

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T-cell mediated immunity can be acquired due to previous exposure to other beta coronaviruses which cause the common cold. Knowing a threshold for herd immunity can allow the government to focus on that section of the population who do not have immunity. But they also caution that very few basic science labs in the country like NIMHANS, IISc, or the National Centre for Biological Sciences can do T cell assays in their labs as it is cumbersome and expensive.

Assessing how much of the population has IgG (non-neutralising antibodies), the current active Covid case burden, and T-cell induced protection, simultaneously will give a clear picture of the health of the population, with respect to Covid-19.

"Currently, we do not know when the pandemic will end. If we know how much of the population is immune, it is easier to decide how much of our resources should be allocated to fight Covid, the economy, etc. If done at the state or the sub-state level, we can understand which region needs more resources," said Dr Giridhar Babu, epidemiologist, and member of the State Covid-19 technical advisory committee (TAC).

In the serosurvey undertaken in Karnataka whose results are yet to be announced, with samples from all the eight zones of Bengaluru included, unlike the serosurveys of Delhi, Mumbai, Pune, and Punjab, Karnataka are supposed to have done all three: RTPCR, antibody, and antigen tests simultaneously in the statewide survey.

Coronavirus India update: State-wise total number of confirmed cases, deaths on October 23

Dr V Ravi, Senior Professor and Head, Neurovirology, NIMHANS, and member of State Covid-19 TAC, told DH, "T cell response assay is very cumbersome and complicated to do. Peripheral blood has to be drawn, lymphocytes separated, culture them, stimulate them with antigens, and then take a readout. It is expensive and resource-intensive. Basic science institutes like IISc, NCBS, National Institute of Immunology, ISER, some of them may have the capacity for doing it, but not the medical college laboratories."

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Covid-19: Has Karnataka achieved herd immunity? Simultaneous triple tests will give true picture - Deccan Herald

Bone Marrow Stem Cells Comments Off on Covid-19: Has Karnataka achieved herd immunity? Simultaneous triple tests will give true picture – Deccan Herald | October 24th, 2020

INTRODUCTION

Cleidocranial dysplasia (CCD) is a hereditary disease characterized by incomplete closure of the fontanelle, abnormal clavicle, short stature, and skeletal dysplasia. It has been reported that there are multiple Runx2 mutations in human CCD syndrome (1, 2). Mature osteoblasts defect and bone mineralization disorders were observed in Runx2-deficient mice. The Runx2-heterozygous mice show similar phenotypes to the CCD syndrome (24). RUNX2 triggers mesenchymal stem cells (MSCs) to differentiate into osteoblasts (3, 5). According to the skeletal pathology studies in humans and mice, it is important to accurately regulate Runx2 activity during bone formation and bone remodeling (6, 7). However, the molecular regulation of Runx2 activity remains to be further studied.

The evolutionarily conserved Hippo pathway is essential for tissue growth, organ size control, and cancer development (811). Numerous evidences revealed the important roles of Hippo components in regulating bone development and bone remodeling. YAP, the essential downstream effector of Hippo pathway, regulates multiple steps of chondrocyte differentiation during skeletal development and bone repair (12). YAP also promotes osteogenesis and suppresses adipogenic differentiation by regulating -catenin signaling (13). VGLL4, a member of the Vestigial-like family, acts as a transcriptional repressor of YAP-TEADs in the Hippo pathway (14). Our previous work found that VGLL4 suppressed lung cancer and gastric cancer progression by directly competing with YAP to bind TEADs through its two TDU (Tondu) domains (9, 15). We also found that VGLL4 played a critical role in heart valve development by regulating heart valve remodeling, maturation, and homeostasis (16). Moreover, our team found that VGLL4 regulated muscle regeneration in YAP-dependent manner at the proliferation stage and YAP-independent manner at the differentiation stage (17). Our previous studies suggest that VGLL4 plays an important role to regulate cell differentiation in multiple organs. However, the function of VGLL4 in skeletal formation and bone remodeling is unknown.

Here, we reveal the function of VGLL4 in osteoblast differentiation and bone development. Our in vivo data show that global knockout of Vgll4 results in a wide variety of skeletal defects similar to Runx2 heterozygote mice. Our in vitro studies reveal that VGLL4 deficiency strongly inhibits osteoblast differentiation. We further demonstrate that TEADs can bind to RUNX2, thereby inhibiting the transcriptional activity of RUNX2 independent of YAP binding. VGLL4 could relieve the inhibitory function of TEADs by breaking its interaction with RUNX2. In addition, deletion of VGLL4 in MSCs shows similar skeletal defects with the global Vgll4-deficient mice. Further studies show that knocking down TEADs or overexpressing RUNX2 in VGLL4-deficient osteoblasts reverses the inhibition of osteoblast differentiation.

To study the function of VGLL4 in bone, we first measured -galactosidase activity in Vgll4LacZ/+ mice (16). -Galactosidase activity was enriched in trabecular bones, cortical bones, cranial suture, and calvaria cultures (fig. S1, A to C). Furthermore, in bone marrow MSCs (BMSCs), Vgll4LacZ/+ mice displayed -galactosidase activity in osteoblast-like cells (fig. S1D). During osteoblast differentiation in vitro, osteoblast marker genes such as alkaline phosphatase (Alp) and Sp7 transcription factor (Osterix) were increased and peaked at day 7. Vgll4 showed similar trend in this process at both mRNA and protein levels (Fig. 1A and fig. S1, E and F). To further clarify the important role of VGLL4 in bone development, we used a Vgll4Vgll4-eGFP/+ reporter mouse line in which VGLL4enhanced green fluorescent protein (eGFP) fusion protein expression is under the control of the endogenous VGLL4 promoter, and GFP staining reflects VGLL4 expression pattern in skeletal tissues (16). GFP staining was performed at embryonic day 18.5, week 1, week 2, and week 4 stages. The results indicated that the VGLL4 expression level was increased during bone development (fig. S1G). In addition, VGLL4 was enriched in trabecular bones, cortical bones, chondrocytes, cranial suture, and calvaria (fig. S1, G and K to M). We then observed the colocalization of VGLL4-eGFP with markers of MSCs (CD105), osteoblasts [osteocalcin (OCN)], and chondrocytes [collagen 2a1 (Col2a1)] in long bone and calvaria (fig. S1, H to M). Next, we analyzed VGLL4 expression pattern during osteoblast development in vivo (fig. S1N), which was similar to Alp and Osterix expression patterns in mouse BMSCs of different ages. Together, both in vivo and in vitro data suggest that VGLL4 may play roles in osteoblast differentiation and bone development.

(A) Immunoblotting showed the expression profile of VGLL4 during osteoblast differentiation in C57BL/6J mouse BMSCs. Samples were collected at 0, 1, 4, 7, and 10 days after differentiation. (B) Skeletons of WT and Vgll4/ mice at postnatal day 1 (P1) were double-stained by Alizarin red/Alcian blue (n = 5). Scale bar, 5 mm. (C) Quantification of body length in (B). (D) Skull preparations from control and Vgll4/ mouse newborns were double-stained with Alizarin red and Alcian blue at P1. -QCT images of skulls were taken from control and Vgll4/ mice at P4. Scale bar, 5 mm. (E) Quantification of skull defect area in (D). (F) Clavicle preparations from control and Vgll4/ mouse newborns were double-stained with Alizarin red and Alcian blue at P1 and quantification of clavicle length. Scale bar, 5 mm. (G) Alp staining and Alizarin red staining of calvarial cells from WT and Vgll4/ mice after cultured in osteogenic medium. Scale bar, 3 mm. (H) Relative mRNA levels were quantified by RT-PCR. (I) Hematoxylin and eosin (H&E) staining of femur from WT and Vgll4/ mice at embryonic day 16.5. Scale bar, 125 m. (J) In situ hybridization for Col11 immunostaining. Scale bar, 125 m. In (C), (E), (F), and (H), data were presented as means SEM; *P < 0.05, **P < 0.01, and ***P < 0.001, ns, no significance; unpaired Students t test. Photo credit: Jinlong Suo, State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai.

To investigate the potential function of VGLL4 in bone, we next analyzed the phenotype of Vgll4 knockout (Vgll4/) mice (16). The newborn Vgll4 knockout mice were significantly smaller and underweight compared with their control littermates (Fig. 1, B and C, and fig. S2, A and B). In particular, the membranous ossification of the skull was impaired in Vgll4/ newborns compared with the control littermates (Fig. 1, D and E). Furthermore, Vgll4 knockout mice developed a marked dwarfism phenotype with short legs and short clavicles (Fig. 1, C and F). To assess the role of VGLL4 in osteoblast differentiation, calvarial cells from Vgll4/ mice and wild-type (WT) mice were cultured in osteogenic medium. The activity of Alp in the Vgll4 deletion group was significantly reduced at the seventh day of differentiation (Fig. 1G, top) and was markedly weakened over a 14-day culture period as revealed by Alizarin red S staining (Fig. 1G, bottom). The declined osteogenesis in Vgll4 knockout cells was confirmed by the decreased expression of a series of osteogenic marker genes (Fig. 1H), including Alp, Osterix, and collagen type1 1 (Col11). In addition, in Vgll4/ mice, bone development was severely impaired with remarkable decrease in bone length and almost a complete loss of bone ossification (Fig. 1I). Consistently, immunohistochemical analysis of bone tissue sections from embryos at embryonic day 14.5 further confirmed the defects of bone formation and impaired osteoblast differentiation in Vgll4/ mice (Fig. 1J). Together, our study suggests that VGLL4 is likely to regulate MSC fate by enhancing osteoblast differentiation.

Given that the smaller size of mice is often caused by dysplasia, we also paid attention to the development of cartilage after Vgll4 deletion. As we expected, cartilage development was delayed in Vgll4-deficient mice determined by Safranin O (SO) staining (fig. S2C). Immunohistochemical analysis of collagen X (Col X) further confirmed the delay of cartilage development in Vgll4/ mice (fig. S2D). However, additional experiments would be required to determine the regulatory mechanism behind the observed chondrodysplasia. Although dwarfism was observed and trabecular bones were significantly reduced in the adult Vgll4/ mice, no significant cartilage disorder was observed by SO staining (fig. S2E). In adults, bone is undergoing continuous bone remodeling, which involves bone formation by osteoblasts and bone resorption by osteoclasts. We speculated that Vgll4 deletion might lead to decreased osteoclast activity. To distinguish this possibility, we performed histological analysis by tartrate-resistant acid phosphatase (TRAP) staining to detect osteoclast activity. The results showed that osteoclast activity was comparable between Vgll4/ mice and their control littermates (fig. S2F). Together, our results suggest that the phenotypes observed in Vgll4/ mice are mainly due to the defect of osteoblast activity.

To further explore the role of Vgll4 in the commitment of MSCs to the fate of osteoblasts, we generated Prx1-cre; Vgll4floxp/floxp mice (hereafter Vgll4prx1 mice) (fig. S3A). Prx1-Cre activity is mainly restricted to limbs and craniofacial mesenchyme cells (18, 19). Western blot analysis confirmed that VGLL4 was knocked out in BMSCs (fig. S3B). Vgll4prx1 mice survived normally after birth and had normal fertility. However, Vgll4prx1 mice exhibited marked dwarfism that was independent of sex (Fig. 2, A and B, and fig. S3C), which was similar to the phenotype of Vgll4/ mice. In particular, the membranous ossification of the skull and clavicle was also impaired in Vgll4prx1 mouse newborns compared with control littermates (Fig. 2, C to E). To assess the role of VGLL4 in osteoblast differentiation, BMSCs from Vgll4prx1 and Vgll4fl/fl mice were cultured in osteogenic medium. Markedly decreased ALP activity and mineralization were observed in Vgll4prx1 mice (Fig. 2, F and G). The declined osteogenesis in Vgll4 knockout osteoblasts was also proved by the decreased expression of a series of osteogenic marker genes, including Alp, Osterix, and Col1a1 (Fig. 2H). Normal Runx2 expression was detected in Vgll4prx1 mice (Fig. 2H). To further verify the role of VGLL4 in osteoblast differentiation, BMSCs from Vgll4fl/fl mice were infected with GFP and Cre recombinase (Cre) lentivirus and then cultured in osteogenic medium. Vgll4fl/fl BMSCs infected with Cre lentivirus showed markedly decreased ALP activity and mineralization (fig. S4A). Reduced VGLL4 expression by Cre lentivirus was confirmed by reverse transcription polymerase chain reaction (RT-PCR) (fig. S4B). The declined osteogenesis was also proved by the decreased expression of a series of osteogenic marker genes, including Alp, Osterix, and Col1a1 (fig. S4B).

(A) Skeletons of Vgll4fl/fl and Vgll4prx1 mice at P1 were double-stained by Alizarin red and Alcian blue. Scale bar, 5 mm. (B) Quantification of body length in (A) (n = 6). (C) Skull and clavicle preparation from Vgll4fl/fl and Vgll4prx1 mouse newborns were double-stained with Alizarin red and Alcian blue at P1. Scale bars, 5 mm. (D) Quantification of the defect area of skulls in (C) (n = 6). (E) Quantification of clavicle length in (C) (n = 6). (F) Alp staining and Alizarin red staining of BMSCs from Vgll4fl/fl and Vgll4prx1 mice after cultured in osteogenic medium. Scale bars, 3 mm. (G) Alp activity was measured by phosphatase substrate assay. (H) Relative mRNA levels were quantified by RT-PCR. (I) 3D -QCT images of trabecular bone (top) and cortical bone (bottom) of distal femurs. (J to N) -QCT analysis for trabecular bone volume per tissue volume (BV/TV, Tb) (J), trabecular number (Tb.N/mm) (K), trabecular thickness (Tb.Th/mm) (L), trabecular separation (Tb.Sp/mm) (M), and cortical bone thickness (Cor.Th/mm) (N). (O) Representative images of von Kossa staining of 12-week-old Vgll4fl/fl and Vgll4prx1 mice. Scale bar, 500 m. (P) Representative images of calcein and Alizarin red S labeling of proximal tibia. Scale bar, 50 m. (Q) Quantification of MAR. (R and S) ELISA analysis of serum PINP (ng ml1) and CTX-1 (ng ml1) from 10-week-old Vgll4fl/fl and Vgll4prx1 mice (n = 5). In (B), (D), (E), (G), (H), (J) to (N), and (Q) to (S), data were presented as means SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; ns, no significance; unpaired Students t test. Photo credit: Jinlong Suo, State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai.

We next performed PCNA (proliferating cell nuclear antigen) staining and MTT assay to detect whether VGLL4 influences cell proliferation during bone development. No significant differences were found after VGLL4 deletion (fig. S5, A to C). We also did not detect significant changes of proliferation-related genes and YAP downstream genes (fig. S5, D and E). We next performed TUNEL (terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick end labeling) staining to detect whether VGLL4 influences cell apoptosis. In addition, no significant differences were found after VGLL4 deletion (fig. S5, F and G).

To further determine the function of VGLL4 in skeletal system, we did micro-quantitative computed tomography (-QCT) analysis to compare the changes in bone-related elements in the long bones of Vgll4prx1 mice and control littermates. We found that the 3-month-old Vgll4prx1 mice showed decreased bone mass per tissue volume (BV/TV) relative to age-matched control littermates (Fig. 2, I and J). Further analysis showed a reduction in trabecular number (Tb.N) of Vgll4prx1 mice compared to control mice (Fig. 2K), which was accompanied by a decrease in trabecular thickness (Tb.Th) and an increase in trabecular separation (Tb.Sp) compared to control mice (Fig. 2, L and M). Vgll4prx1 mice also showed decreased cortical bone thickness (Cor.Th) relative to the Vgll4fl/fl mice (Fig. 2N). The von Kossa staining showed reduced bone mineral deposition in 3-month-old Vgll4prx1 mice (Fig. 2O). The mineral apposition rate (MAR) was also decreased in Vgll4prx1 mice compared with control littermates by fluorescent double labeling of the mineralizing front (Fig. 2, P and Q). Consistent with the decreased bone mass in Vgll4prx1 mice, the enzyme-linked immunosorbent assay (ELISA) assay of N-terminal propeptide of type I procollagen (PINP), a marker of bone formation, revealed a reduced bone formation rate in Vgll4prx1 mice (Fig. 2R). However, the ELISA assay of C-terminal telopeptide of collagen type 1 (CTX-1), a marker of bone resorption, showed that the bone resorption rate of Vgll4prx1 mice did not change significantly (Fig. 2S). Collectively, Vgll4 conditional knockout mice mimicked the main phenotypes of the global Vgll4 knockout mice, further indicating that VGLL4 specifically regulates bone mass by promoting osteoblast differentiation.

To further determine whether the abnormal osteogenesis in Vgll4prx1 mice was caused by a primary defect in osteoblast development, we generated an osteoblast-specific Osx-cre; Vgll4floxp/floxp mice (hereafter Vgll4Osx mice) by crossing Vgll4fl/fl mice with Osx-Cre mice, a line in which Cre expression is primarily restricted to osteoblast precursors (fig. S6A) (6, 20). Vgll4Osx mice survived normally after birth and had normal fertility, but exhibited marked dwarfism in comparison with Osx-Cre mice (fig. S6, B and C), which was similar to the phenotypes of Vgll4/ and Vgll4prx1 mice. In addition, the membranous ossification of the skull and clavicle was also impaired in Vgll4Osx mice compared with control littermates (fig. S6C). -QCT analysis further confirmed the osteogenic phenotype of Vgll4Osx mice (fig. S6, D to J). Hence, the Vgll4Osx mice summarized the defects observed in the Vgll4prx1 mice, thus supporting the conclusion that VGLL4 is necessary for the differentiation and function of committed osteoblast precursors.

We next worked to figure out the mechanism how VGLL4 controls bone mass and osteoblast differentiation. The pygmy and cranial closure disorders in Vgll4/ mice were similar to that of Runx2-heterozygous mice. We therefore examined the potential interaction between VGLL4 and RUNX2. However, coimmunoprecipitation experiments did not show the interaction between VGLL4 and RUNX2 (Fig. 3A). Previous studies showed that VGLL4 could compete with YAP for binding to TEADs (9). The TEAD family contains four highly homologous proteins (8), which is involved in the regulation of myoblast differentiation and muscle regeneration (21). We determined whether the binding of VGLL4 with RUNX2 requires TEADs. Coimmunoprecipitation experiments showed that RUNX2 and TEAD14 had almost equivalent interactions (Fig. 3B). Next, we investigated whether TEADs control the transcriptional activity of Runx2. We used the 6xOSE2-luciferase reporter system that is specifically activated by RUNX2 to verify the role of TEADs (22). We performed dual-luciferase reporter assay with 6xOSE2-luciferase and Renilla in C3H10T1/2 cells, and the results showed that TEAD14 significantly inhibited the activation of 6xOSE2-luciferase induced by RUNX2 (Fig. 3C). Consistently, knockdown of TEADs by small interfering RNAs (siRNAs) markedly enhanced both basic and RUNX2-induced 6xOSE2-luciferase activity (fig. S8A). TEAD family is highly conserved, which consists of an N-terminal TEA domain and a C-terminal YAP-binding domain (YBD) (Fig. 3D) (23). Glutathione S-transferase (GST) pull-down assay revealed the direct interaction between RUNX2 and TEAD4 (Fig. 3E). Moreover, both TEA and YBD domains of TEAD4 could bind to RUNX2 (Fig. 3, F and G).

(A) Coimmunoprecipitation experiments of RUNX2 and VGLL4 in HEK-293T cells. The arrow indicated IgG heavy chain. (B) Coimmunoprecipitation experiments of RUNX2 and TEAD14 in HEK-293T cells. The arrow indicated IgG heavy chain. (C) 6xOSE2-luciferase activity was determined in C3H10T1/2 cells cotransfected with RUNX2 and TEAD14. Data were calculated from three independent replicates. (D) Schematic illustration of the domain organization for TEAD4, TEAD4-Nt, and TEAD4-Ct. (E) GST pull-down (PD) analysis between purified GST-RUNX2 and HIS-SUMO-TEAD4 proteins. (F) GST pull-down analysis between purified GST-RUNX2 and HIS-SUMO-TEAD4-TEA proteins. (G) Lysates from HEK-293T cells with Flag and Flag-RUNX2 expressions were incubated with recombinant GST-TEAD4-YBD protein. GST pull-down assay showed the binding between RUNX2 and TEAD4-YBD. (H) Cells isolated from WT mice were infected with TEAD lentivirus. Osteoblast differentiation was evaluated by Alp staining and Alizarin red staining after culture in osteoblast differentiation medium for 7 days (top) and 14 days (bottom). Data are representative of three independent experiments. Scale bars, 3 mm. (I) Alp activity quantification was measured by phosphatase substrate assay (n = 3). (J) Relative mRNA levels of Alp, Col11, and Osterix were quantified by RT-PCR. (K) Cells isolated from WT mice were infected with TEAD shRNA lentivirus. Osteoblast differentiation was evaluated by Alp staining and Alizarin red staining after culture in osteoblast differentiation medium for 7 days (top) and 14 days (bottom). Data are representative of three independent experiments. Scale bars, 3 mm. (L) Alp activity quantification was measured by phosphatase substrate assay (n = 3). (M) Relative mRNA levels of Runx2, Alp, Col11, and Osterix were quantified by RT-PCR. (N) Relative mRNA levels of Tead1-4 were quantified by RT-PCR. In (C), (I), (J), and (L) to (N), data were presented as means SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; ns, no significance; unpaired Students t test.

To determine whether overexpression of TEAD14 affects osteoblast differentiation, BMSCs from WT mice were infected with TEAD14 lentivirus and then cultured in osteogenic medium. The activities of ALP in TEAD14 overexpression groups were significantly reduced at the seventh day of differentiation [Fig. 3, H (top) and I] and were significantly weakened by Alizarin red S staining over a 14-day culture period (Fig. 3H, bottom). The declined osteogenesis in TEAD14 overexpression cells was confirmed again by the decreased expression of a series of osteogenic marker genes, including Alp, Col11, and Osterix (Fig. 3J). Next, we blocked the total activities of TEAD14 by short hairpin RNA (shRNA) lentiviral infection (Fig. 3N). The activity of Alp in TEAD14 knockdown group was significantly increased [Fig. 3, K (top) and L]. Over a 14-day culture period, osteogenic differentiation was significantly enhanced by Alizarin red S staining (Fig. 3K, bottom). The enhanced osteogenesis in TEAD14 knockdown cells was further confirmed by elevated expression of a series of osteogenic marker genes, including Alp, Col11, and Osterix (Fig. 3M). These results suggest that TEAD14 act as repressors of RUNX2 to inhibit osteoblast differentiation.

To investigate the mechanistic role of VGLL4 in inhibiting osteoblast differentiation, we then verified whether VGLL4 could affect the interaction between TEADs and RUNX2. We found that VGLL4 reduced the interaction between RUNX2 and TEADs (Fig. 4A). To further illustrate the relationship between RUNX2/TEADs/VGLL4, we checked the interaction between RUNX2 and TEADs in the BMSC of Vgll4fl/fl mice treated with GFP or Cre lentivirus. We found that the interaction between RUNX2 and TEADs was enhanced in Cre-treated cells (Fig. 4B). We noticed that there were conserved binding sites of RUNX2 (5-AACCAC-3) and TEAD (5-CATTCC-3) in the promoter regions of Alpi, Osx, and Col1a1, which are three target genes of RUNX2 (17, 24). We performed TEAD4 and RUNX2 chromatin immunoprecipitation (ChIP) assays in BMSCs. The results indicated that both TEAD4 and RUNX2 bound on Alp, Osx, and Col1a1 promoters (fig. S7, A to I). VGLL4 was a transcriptional cofactor, which could not bind DNA directly. We have demonstrated that VGLL4 promoted RUNX2 activity by competing for its binding to TEADs. Consistently, VGLL4 partially blocked TEADs-repressed transcriptional activity of RUNX2 (Fig. 4C). However, overexpression of VGLL4 in TEADs knockdown cells showed no marked change on RUNX2-induced 6xOSE2-luciferase activity compared with TEAD knockdown (fig. S8B). We then asked whether loss of VGLL4-induced disorders of osteoblast differentiation is related to TEADs. We knocked down TEADs by lentiviral infection in Vgll4-deficient BMSCs and then induced these cells for osteogenic differentiation. The differentiation disorders caused by VGLL4 deletion were restored after TEAD knockdown (Fig. 4, D to F). These data supported that VGLL4 released the inhibition of TEADs on RUNX2, thereby promoting osteoblast differentiation.

(A) Coimmunoprecipitation experiments of RUNX2, TEADs, and VGLL4 in HEK-293T cells. The arrow indicated IgG heavy chain. (B) Coimmunoprecipitation experiments of RUNX2 and TEADs in BMSCs cells of Vgll4fl/fl mice treated with GFP and Cre lentivirus. (C) 6xOSE2-luciferase activity was determined in C3H10T1/2 cells cotransfected with RUNX2, TEADs, and VGLL4. (D) Cells isolated from Vgll4fl/fl and Vgll4prx1 mice were infected with GFP and TEAD shRNA lentivirus. Osteoblast differentiation was evaluated by Alp staining and Alizarin red staining after culture in osteoblast differentiation medium for 7 days (top) and 14 days (bottom). Data are representative of three independent experiments. Scale bars, 3 mm. (E) Alp activity quantification was measured by phosphatase substrate assay (n = 3). (F) Relative mRNA levels of Vgll4, Runx2, Alp, Col11, and Osterix were quantified by RT-PCR. In (B), (D), and (E), data were presented as means SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; ns, no significance; unpaired Students t test.

YAP, the key transcription cofactor in the Hippo pathway, has been widely reported in regulating bone development and bone mass (12, 13). VGLL4, a previously identified YAP antagonist, directly competes with YAP for binding to TEADs (9). Therefore, we suspected that the inhibition of RUNX2 transcriptional activity caused by VGLL4 deletion might be dependent on YAP. To this end, we validated the role of YAP by 6xOSE2-luciferase reporter system. The data showed that YAP promoted RUNX2 activity in a dose-dependent manner (Fig. 5A). Moreover, TEAD4 significantly inhibited 6xOSE2-luciferase activity induced by YAP (Fig. 5B). TEAD4Y429H, a mutation that impairs the interaction between TEAD4 and YAP/TAZ (Fig. 5C) (25), did not promote 3xSd-luciferase activity induced by YAP (Fig. 5D). We found that both TEAD and TEAD4Y429H could interact with RUNX2 (Fig. 5E), and both TEAD4 and TEAD4Y429H could inhibit the activity of RUNX2 in a dose-dependent manner (Fig. 5, F and G). Restoring the expression of both TEAD4 and TEAD4Y429H could reverse the increased osteoblast differentiation in TEAD knockdown BMSCs (Fig. 5, H and I). Furthermore, overexpression of TEAD1 could further inhibit osteogenic differentiation of BMSCs after YAP knockdown (Fig. 5J). Together, these data suggest that the inhibition of RUNX2 activity by TEADs is independent of YAP binding.

(A) Effects of YAP on Runx2-activated 6xOSE2-luciferase activity in C3H10T1/2 cells. (B) 6xOSE2-luciferase activity was determined in C3H10T1/2 cells cotransfected with RUNX2, YAP, and TEAD4. (C) Schematic illustration of TEAD4 and TEAD4Y429H mutation. (D) 3xSd-luciferase activity was determined in HEK-293T cells cotransfected with YAP, TEAD4, and TEAD4Y429H. (E) Coimmunoprecipitation experiments of RUNX2, TEAD4, and TEAD4Y429H in HEK-293T cells. The arrow indicated IgG heavy chain. (F) Effects of TEAD4 on RUNX2-activated 6xOSE2-luciferase activity in C3H10T1/2 cells. (G) Effects of TEAD4Y429H on RUNX2-activated 6xOSE2-luciferase activity in C3H10T1/2 cells. (H) Cells isolated from WT mice were infected with GFP or TEAD shRNAs, TEAD4, or TEAD4Y429H lentivirus. Osteoblast differentiation was evaluated by Alp staining and Alizarin red staining after culture in osteoblast differentiation medium for 7 days (top) and 14 days (bottom). Data are representative of three independent experiments. Scale bars, 3 mm. (I) Alp activity quantification was measured by phosphatase substrate assay (n = 3). (J) Relative mRNA levels of Runx2, Alp, Col11, Osterix, Tead1, and Yap were quantified by RT-PCR. In (A), (B), (D), (F), (G), (I), and (J), data were presented as means SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; ns, no significance; unpaired Students t test.

We next examined how VGLL4 breaks the interaction between RUNX2 and TEADs. It has been reported that VGLL4 relies on its own two TDU domains to interact with TEADs (9), and VGLL4 HF4A mutation can disrupt the interaction between VGLL4 and TEADs (15). We hypothesized that VGLL4 competes with RUNX2 for TEAD1 binding depending on its TDU domain. On the basis of these previous studies, we performed coimmunoprecipitation experiments and found that VGLL4 HF4A abolished the interaction between VGLL4 and TEAD1 but did not affect the interaction between TEAD1 and RUNX2 (Fig. 6A). VGLL4 partially rescued the inhibition of RUNX2 transcriptional activity by TEAD1; however, VGLL4 HF4A lost this function (Fig. 6B). We then overexpressed TEAD1 by lentivirus infection in primary calvarial cells and found that the transcriptional level of Alp was significantly inhibited. This inhibition was released by overexpressing VGLL4 but not VGLL4 HF4A (Fig. 6C). To further verify the specific regulation of RUNX2 activity by VGLL4, we performed a coimmunoprecipitation experiment with low and high doses of VGLL4 and VGLL4 HF4A. The results showed that the TEAD1-RUNX2 interaction was gradually repressed along with an increasing dose of VGLL4 but not VGLL4 HF4A (Fig. 6D). Similarly, the inhibition of RUNX2 transcriptional activity by TEAD1 was gradually released with an increasing dose of VGLL4 but not VGLL4 HF4A (Fig. 6E). Super-TDU, a peptide mimicking VGLL4, could also reduce the interaction between purified RUNX2 and TEAD4 proteins (Fig. 6F). Thus, these findings suggest that VGLL4 TDU domain competes with RUNX2 for TEADs binding to release RUNX2 transcriptional activity.

(A) Coimmunoprecipitation experiments of RUNX2, TEAD1, VGLL4, and VGLL4 HF4A in HEK-293T cells. The arrow indicated IgG heavy chain. (B) 6xOSE2-luciferase activity was determined in C3H10T1/2 cells cotransfected with RUNX2, VGLL4, VGLL4 HF4A, and TEAD1 (n = 3). (C) RT-PCR analysis of Alp expression in calvarial cells. Cells isolated from WT mice were infected with GFP, TEAD1, VGLL4, or VGLL4 HF4A lentivirus. (D) Coimmunoprecipitation experiments of RUNX2, TEAD1, and an increasing amount of VGLL4 or VGLL4 HF4A in HEK-293T cells. The arrow indicated IgG heavy chain. (E) 6xOSE2-luciferase activity was determined in C3H10T1/2 cells cotransfected with RUNX2, TEAD1, and an increasing amount of VGLL4 or VGLL4 HF4A. (F) Competitive GST pull-down assay to detect the effect of VGLL4 Super-TDU on the interaction between RUNX2 and TEAD4. (G) Cells isolated from Vgll4fl/fl and Vgll4prx1 mice were infected with GFP and RUNX2 lentivirus. Osteoblast differentiation was evaluated by Alp staining and Alizarin red staining after culture in osteoblast differentiation medium for 7 days (top) and 14 days (bottom). Data are representative of three independent experiments. Scale bars, 3 mm. (H) Alp activity quantification was measured by phosphatase substrate assay (n = 3). (I) Relative mRNA levels of Vgll4, Runx2, Alp, Col11, and Osterix were quantified by RT-PCR. (J) Schematic model of VGLL4/TEADs/RUNX2 in regulating osteogenic differentiation. In (B), (C), (E), (H), and (I), data were presented as means SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; ns, no significance; unpaired Students t test.

Furthermore, we overexpressed RUNX2 by lentivirus infection in Vgll4 knockout BMSCs during osteogenic differentiation, and we found that RUNX2 could significantly restore the osteogenic differentiation disorder caused by Vgll4 deletion (Fig. 6, G to I). Together, these data suggest a genetic interaction between VGLL4/TEADs/RUNX2 and provide evidences that RUNX2 overexpression rescues osteogenic differentiation disorders caused by VGLL4 deletion.

Collectively, our study demonstrates the important roles of VGLL4 in osteoblast differentiation, bone development, and bone homeostasis. In the early stage of osteoblast differentiation, TEADs interact with RUNX2 to inhibit its transcriptional activity in a YAP bindingindependent manner. During differentiation progress, VGLL4 expression gradually increases to dissociate the interaction between TEADs and RUNX2, thereby releasing the inhibition of RUNX2 transcriptional activity by TEADs and promoting osteoblasts differentiation (Fig. 6J).

Accumulating evidences have suggested that the Hippo pathway plays key roles in regulating organ size and tissue homeostasis (8, 10). However, the transcription factors TEADs have not been reported in skeletal development and bone-related diseases. VGLL4 functions as a new tumor suppressor gene, which has been reported to negatively regulate the YAP-TEADs transcriptional complex. Our previous studies show that VGLL4 plays important roles in many tissue homeostasis and organ development, such as heart and muscle (16, 17). In this study, we provide evidences to show that VGLL4 can break TEADs-mediated transcriptional inhibition of RUNX2 to promote osteoblast differentiation and bone development independent of YAP binding.

Overall, our studies establish the Vgll4-specific knockout mouse model in the skeletal system. We show that VGLL4 deletion in MSCs leads to abnormal osteogenic differentiation with delayed skull closure and reduced bone mass. Our data also reveal that VGLL4 deletion leads to chondrodysplasia. Recent researches identified that chondrocytes have the ability to transdifferentiate into osteoblasts (2628), suggesting the possibility that loss of VGLL4 might reduce or delay the pool of chondrocytes that differentiate into osteoblasts. We identify that VGLL4 regulates the RUNX2-TEADs transcriptional complex to control osteoblast differentiation and bone development. TEADs can bind to RUNX2 and inhibit its transcriptional activity in a YAP bindingindependent manner. Recent studies pointed out that reciprocal stabilization of ABL and TAZ regulates osteoblastogenesis through transcription factor RUNX2 (29); however, we found that TEAD4-Y429H, a mutation at the binding site of TAZ and TEAD (25, 30, 31), can still significantly inhibit the activity of RUNX2. Therefore, we consider that the way TEAD regulates RUNX2 may not depend on TAZ regulation. Further research found that VGLL4, but not VGLL4 HF4A, can alleviate the inhibition by influencing the binding between RUNX2 and TEADs. It is possible that VGLL4 might influence the structure organization of the RUNX2-TEAD complex to some extent. Structural information may be required to answer this question and may provide more insights into the mechanism of VGLL4 in osteogenic differentiation.

Previous studies showed that mutations in RUNX2 cause CCD and Runx2+/ mice show a CCD-like phenotype. However, many patients with CCD do not have RUNX2 mutations. Our study may provide clues to the pathogenesis of these patients. A significant reduction of bone mass was observed in the adult mice, suggesting that VGLL4 and TEADs might be drug targets for treatment of cranial closure disorders and osteoporosis. In addition, further investigation of the clinical correlation of VGLL4 and cleidocranial dysplasia in a larger cohort will provide more accurate information for bone research. Our work also provides clues to researchers who are studying the roles of VGLL4 in tumors or other diseases. RUNX2 is highly expressed in breast and prostate cancer cells. RUNX2 contributes to tumor growth in bone and the accompanying osteolytic diseases (32). The regulation of RUNX2 transcriptional activity by TEADs and VGLL4 is likely to play essential roles in tumor, bone metastasis, and osteolytic diseases. Our work may provide clues to researchers who are studying the role of VGLL4 in bone tumors.

We demonstrate that TEADs are involved in regulating osteoblast differentiation by overexpressing and knocking down the TEAD family in vitro. However, the exact roles of TEADs in vivo need to be further confirmed by generation of TEAD1/2/3/4 conditional knockout mice. In the follow-up work, we will continue to study the mechanism of TEADs in skeletal development and bone diseases. Overall, although there are still some shortcomings, our work has greatly contributed to understand the TEADs regulation of RUNX2 activity.

Our work defines the role of VGLL4 in regulating osteoblast differentiation and bone development, and identifies that TEADs function as repressors of RUNX2 to inhibit osteoblast differentiation. We propose a model that VGLL4 dissociates the combination between TEADs and RUNX2. It is not clear whether VGLL4 is also involved in regulating other transcription factors or signaling pathways in the process of osteoblast differentiation and bone development. If that is the case, how to achieve cooperation will be another interesting issue worthy of further study.

Vgll4Lacz/+ mice, Vgll4 knockout (Vgll4/) mice, Vgll4Vgll4-eGFP/+ mice, and Vgll4 conditional knockout (Vgll4fl/fl) mice were generated as previously described (16, 17), and Vgll4fl/fl mice were crossed with the Prx1-Cre and Osx-Cre strain to generate Vgll4prx1 and Vgll4Osx mice. All mice analyzed were maintained on the C57BL/6 background. All mice were monitored in a specific pathogenfree environment and treated in strict accordance with protocols approved by the Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

The following antibodies were used: anti-Osterix antibody (1:1000; Santa Cruz Biotechnology, SC133871), anti-RUNX2 antibodies (1:1000; Santa Cruz Biotechnology, SC-390351 and SC-10758), anti-Flag antibody (1:5000; Sigma-Aldrich, F-3165), anti-HA (hemagglutinin) antibody (1:2000; Santa Cruz Biotechnology, SC-7392), anti-HA antibody (1:1000; Sangon Biotech, D110004), anti-MYC antibody (1:1000; ABclonal Technology, AE010), anti-PCNA antibody (1:1000; Santa Cruz Biotechnology, SC-56), rabbit immunoglobulin G (IgG) (Santa Cruz Biotechnology, SC-2027), mouse IgG (Sigma-Aldrich, I5381), anti-VGLL4 antibody (1:1000; ABclonal, A18248), anti-TEAD1 antibody (1:1000; ABclonal, A6768), anti-TEAD2 antibody (1:1000; ABclonal, A15594), anti-TEAD3 antibody (1:1000; ABclonal, A7454), anti-TEAD4 antibody (1:1000; Abcam, ab58310), and antipan-TEAD (1:1000; Cell Signaling Technology, 13295).

Cells were cultured at 37C in humidified incubators containing an atmosphere of 5% CO2. Human embryonic kidney (HEK)293T cells were maintained in Dulbeccos Modified Eagle Medium (DMEM) (Corning, Corning, NY) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco) solution. C3H10T1/2 cells were maintained in -minimum essential medium (-MEM) (Corning, Corning, NY) supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco) solution. To induce differentiation of BMSC into osteoblasts, cells were cultured in -MEM containing 10% FBS, l-ascorbic acid (50 g/ml), and -glycerophosphate (1080 mg/ml). The osteoblast differentiation level assay was performed following a previously published method (33). To quantitate Alp activity, cells incubated with Alamar Blue to calculate cell numbers and then incubated with phosphatase substrate (Sigma-Aldrich, St. Louis, MO) dissolved in 6.5 mM Na2CO3, 18.5 mM NaHCO3, and 2 mM MgCl2 after washing by phosphate-buffered saline (PBS). Alp activity was then read with a luminometer (Envision). Bone nodule formation was stained with Alizarin red S solution (1 mg/ml; pH 5.5) after 14 days of induction.

We collected femurs and tibias from mice and flushed out the bone marrow cells with 10% FBS in PBS. All nuclear cells were seeded (2 106 cells per dish) in 100-mm culture dishes (Corning) and incubated at 37C under 5% CO2 conditions. After 48 hours, nonadherent cells were washed by PBS and adherent cells were cultured in -MEM (Corning, Corning, NY) supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco) solution for an additional 5 days. Mouse BMSCs in passage one were used in this study.

Total RNA was isolated from cells with TRIzol reagent (T9424, Sigma-Aldrich), and first-strand complementary DNA (cDNA) was synthesized from 0.5 g of total RNA using the PrimeScript RT Reagent Kit (PR037A, TaKaRa). The real-time RT-PCR was performed with the Bio-Rad CFX96 System. Gene expression analysis from RT-PCR was quantified relative to Hprt.

C3H10T1/2 cells were seeded overnight at 1 105 cells per well into a 12-well plate and transfected by PEI (polyethylenimine linear) with a luciferase reporter plasmid along with various expression constructs, as indicated. All wells were supplemented with control empty expression vector plasmids to keep the total amount of DNA constant. At 36 to 48 hours after transfection, the cells were harvested and subjected to dual-luciferase reporter assays according to the manufacturers protocol (Promega).

293T cells were seeded at 1 107 cells per 10-cm dish and cultured overnight. At 36 to 48 hours after transfection with PEI, cells were harvested and washed with cold PBS following experimental treatments. Then, cells were lysed with EBC buffer [50 mM tris (pH 7.5), 120 mM NaCl, and 0.5% NP-40] containing protease inhibitor cocktail (1:100; MedChem Express, HY-K0010). After ultrasonication, lysates were subjected to immunoprecipitation with anti-Flag antibodies (M2, Sigma-Aldrich) at 4C overnight, followed by washing in lysis buffer, SDSpolyacrylamide gel electrophoresis (PAGE), and immunoblotting with the indicated antibody.

RUNX2 and TEAD4-YBD were cloned into pGEX-4T-1-GST vector and expressed in Escherichia coli BL21 (DE3) cells. TEAD4 and TEAD4-TEA were cloned into HT-pET-28a-HIS-SUMO vector and expressed in E. coli BL21 (DE3) cells. The two TDU domains of VGLL4 were cloned into HT-pET-28a-MBP vector and expressed in E. coli BL21 (DE3) cells. VGLL4 Super-TDU was designed as previously described (15). GST, HIS-SUMO, and MBP-fused proteins were purified by affinity chromatography as previously described (17). The input and output samples were loaded to SDS-PAGE and detected by Western blotting.

CalceinAlizarin red S labeling measuring bone formation rate was performed as previously described (33).

Preparation of skeletal tissue and -QCT analysis were performed as previously described (34). The mouse femurs isolated from age- and sex-matched mice were skinned and fixed in 70% ethanol. Scanning was performed with the -QCT SkyScan 1176 System (Bruker Biospin). The mouse femurs were scanned at a 9-m resolution for quantitative analysis. Three-dimensional (3D) images were reconstructed using a fixed threshold.

ChIP experiments were carried out in BMSCs according to a standard protocol. The cell lysate was sonicated for 20 min (30 s on, 30 s off), and chromatin was divided into fragments ranging mainly from 200 to 500 base pairs in length. Immunoprecipitation was then performed using antibodies against TEAD4 (Abcam, ab58310), RUNX2 (Santa Cruz Biotechnology, SC-10758), and normal IgG. The DNA immunoprecipitated by the antibodies was detected by RT-PCR. The primers used were as follows: Alp-OSE2-ChIP-qPCR-F (5-GTCTCCTGCCTGTGTTTCCACAGTG-3), Alp-OSE2-ChIP-qPCR-R (5-GAAGACGCCTGCTCTGTGGACTAGAG-3), Alp-TBS-ChIP-qPCR-F (5-CCTTGCATGTAAATGGTGGACATGG-3), Alp-TBS-ChIP-qPCR-R (5-TATCATAGTCACTGAGCACTCTCTTGCG-3), Osx-OSE2-ChIP-qPCR-F (5-TTAACTGCCAAGCCATCGCTCAAG-3), Osx-OSE2-ChIP-qPCR-R (5-CCTCTATGTGTGTATGTGTGTTTACCAAACATC-3), Osx-TBS-ChIP-qPCR-F (5-ATGCCAAGAGATCCCTCATTAGGGAC-3), Osx-TBS-ChIP-qPCR-R (5-AGCTTGGTGAGCACAGCAAAGACAC-3), Col1a1-TBS/OSE2-Chip-qPCR-F (5-CTCAGCCTCAGAGCTGTTATTTATTAGAAAGG-3), and Col1a1-TBS/OSE2-Chip-qPCR-R (5-TTAATCTGATTAGAACCTATCAGCTAAGCAGATG-3). TBS indicated TEAD binding sites.

Mouse TEAD1, TEAD2, TEAD3, and TEAD4 siRNAs and the control siRNA were synthesized from Shanghai Gene Pharma Co. Ltd., Shanghai, China. siRNA oligonucleotides were transfected in C3H10T1/2 by Lipofectamine RNAiMAX (Invitrogen) following the manufacturers instructions. Two pairs of siRNAs were used to perform experiments.

Hematoxylin and eosin stain and immunohistochemistry were performed as previously described (7). Tissue sections were used for TRAP staining according to the standard protocol. Tissues were fixed in 4% paraformaldehyde for 48 hours and incubated in 15% DEPC (diethyl pyrocarbonate)EDTA (pH 7.8) for decalcification. Then, specimens were embedded in paraffin and sectioned at 7 m. Immunofluorescence was performed as previously described (33). Sections were blocked in PBS with 10% horse serum and 0.1% Triton for 1 hour and then stained overnight with anti-PCNA antibody (SC-56). Donkey anti-rabbit Alexa Fluor 488 (1:1000; Molecular Probes, A21206) was used as secondary antibodies. DAPI (4,6-diamidino-2-phenylindole) (Sigma-Aldrich, D8417) was used for counterstaining. Slides were mounted with anti-fluorescence mounting medium (Dako, S3023), and images were acquired with a Leica SP5 and SP8 confocal microscope. For embryonic mice, 5-mm tissue sections were used for immunohistochemistry staining, DIG-labeled in situ hybridization (Roche), and immunohistochemical staining (Dako).

TUNEL staining for apoptosis testing was performed as provided by Promega (G3250).

MTT assay for cell viability was performed as provided by Thermo Fisher Scientific.

We determined serum concentrations of PINP using the Mouse PINP EIA Kit (YX-160930M) according to the instructions provided. In addition, we determined serum concentrations of CTX-1 using the Mouse CTX-1 EIA Kit (YX-032033M) according to the instructions provided.

Tissue sections were used for SO staining according to the standard protocol. After paraffin sections were dewaxed into water, they were acidified with 1% acetic acid for 10 s and then fast green for 2 min, acidified with 1% acetic acid for 10 s, stained with SO for 3 min and 95% ethanol for 5 s, and dried and sealed with neutral glue.

Statistical analysis was performed by unpaired, two-tailed Students t test for comparison between two groups using GraphPad Prism Software. A P value of less than 0.05 was considered statistically significant.

Acknowledgments: We thank A. McMahon (Harvard University, Boston, MA) for providing the Prx1-Cre mouse line. We thank the cell biology core facility and the animal core facility of Shanghai Institute of Biochemistry and Cell Biology for assistance. Funding: This work was supported by the National Natural Science Foundation of China (nos. 81725010, 31625017, 81672119, and 31530043), National Key Research and Development Program of China (2017YFA0103601 and 2019YFA0802001), Strategic Priority Research Program of Chinese Academy of Sciences (XDB19000000), Shanghai Leading Talents Program, Science and Technology Commission of Shanghai Municipality (19ZR1466300), and Youth Innovation Promotion Association CAS (2018004). Author contributions: Z.W., L.Z., and W.Z. conceived and supervised the study. J.S. conceived and designed the study, performed the experiments, analyzed the data, and wrote the manuscript. X.F. made the constructs, performed the in vitro pull-down assay and ChIP experiments, analyzed the data, and revised the manuscript. L.Z. and Z.W. provided genetic strains of mice. J.S. and Z.W. bred and analyzed Vgll4/ mice. J.L. and J.W. cultured the cells and made the constructs. W.Z., L.Z., X.F., and Z.W. edited the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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VGLL4 promotes osteoblast differentiation by antagonizing TEADs-inhibited Runx2 transcription - Science Advances

Bone Marrow Stem Cells Comments Off on VGLL4 promotes osteoblast differentiation by antagonizing TEADs-inhibited Runx2 transcription – Science Advances | October 24th, 2020

INTRODUCTION

Ewing sarcoma (EwS) is an aggressive, poorly differentiated, human tumor characterized by a chromosomal translocation involving a member of the FET family of genes (FUS, EWSR1 and TAF15) and a member of the ETS family of transcription factors, with the EWSR1-FLI1 gene fusion the most common one (1). EwS genomes present low mutation rates with FET-ETS rearrangements as the dominant genetic aberration in the majority of tumors (2). Notably, the cell of origin of EwS is still a controversial field, although human mesenchymal stem cells (hMSCs) and human neural crest stem cells are the most accepted (35).

The EWSR1-FLI1 fusion protein, which contains the transcriptional activation and RNA binding domains of EWSR1 and the DNA binding domain of FLI1, is the main driver of tumorigenesis (3, 6). The resulting fusion oncoprotein has the ability to act as an aberrant transcription factor, leading to gene activation and repression for a well-described set of genes (3, 7). A decade ago, EWSR1-FLI1 was found to bind preferentially to DNA sites containing GGAA microsatellite repeats (8, 9). Recent studies have reported that binding of EWSR1-FLI1 multimers to GGAA repeats acts as a pioneer factor and induces the formation of de novo active enhancers by recruiting the acetyl transferases CBP/p300, E2F3, and the BRG1/BRM-associated factor chromatin remodeling complex (1012). On the other hand, it was hypothesized that monomeric EWSR1-FLI1 inhibits transcription at enhancers by displacing endogenous ETS transcription factors from GGAA motifs (10). Therefore, the mechanisms by which EWSR1-FLI1 acts as either a gene activator or repressor depend on both DNA sequence and cofactors.

Several proteins from the Polycomb group (PcG) have previously been implicated in EwS tumorigenesis. PcG was first described in Drosophila melanogaster as a key regulator of Hox genes expression. PcG proteins not only prevent differentiation by repressing lineage-specific genes but also mark bivalent chromatin regions for subsequent activation. EZH2 (the enzymatic subunit of PRC2) methylates histone H3 at lysine 27 (H3K27me3), while RING1B (the enzymatic subunit of PRC1) ubiquitinates H2A at lysine 119 (H2Aub), both considered repressive histone marks (13).

The canonical PRC1 complex (defined by the presence of four subunits, comprising one variant each of PCGF, PHC, CBX, and RING1) has mostly been associated with maintaining gene repression. However, increasing evidence indicates that PRC1 complexes containing RING1B have the potential for transcription activation, via their catalytic-independent association with UTX, an H3K27me3 demethylase, and p300 acetyltransferase (14, 15). With respect to EwS, it was recently shown that EZH2 blocks endothelial and neuroectodermal differentiation (16), BMI1 promotes tumorigenicity (17), and RING1B represses the nuclear factor B pathway (18). The molecular mechanisms behind the contribution of PcG to EwS have not been addressed. Notably, the GGAA repeats are significantly decorated with H3K27me3 in H1 human embryonic cell lines and human umbilical vein endothelial cells (HUVECs) (19). This is in stark contrast with the lack of H3K27me3 mark at EWSR1-FLI1 binding sites in EwS cells (10, 11), thus suggesting a different role of PcG in EwS. Last, comparison between malignant and nonmalignant tissues revealed a misregulation of PcG target genes in EwS (20). Together, these findings suggest a potential role of the PcG during the early steps of EwS pathogenesis. Here, we report that RING1B and EWSR1-FLI1 interact and colocalize at the same genomic loci. Notably, we find that RING1B is present at promoters and enhancers of actively transcribed EWSR1-FLI1 target genes. Furthermore, we demonstrate that modulation of RING1B interferes with EWSR1-FLI1 recruitment and with the expression of EWSR1-FLI1 targets, thus unveiling an interdependent cooperation between both proteins.

Human pediatric MSCs (hpMSCs) have been proposed as a plausible cell of origin for EwS (21). Nevertheless primary human endothelial HUVECs share high similarity in gene expression profiles with EwS cells (22). Thus, to investigate the potential contribution of epigenetic alteration in the initiation of EwS, we analyzed the role of epigenetic marks in these models and compared to established EwS cell lines. We first analyzed the levels of H3K27me3 and H3K4me3 in the human EwS-derived cell line A673 at several bona fide direct targets of EWSR1-FLI1 (table S1) by chromatin immunoprecipitation followed by quantitative polymerase chain reaction (ChIP-qPCR). Promoter of genes that are transcriptionally activated by EWSR1-FLI1, such as FCGRT, NR0B1, CACNB2, EZH2, IGF1, NKX2-2, and HOXD11, was enriched for the H3K4me3 mark, and lacked the H3K27me3 mark, in agreement with previous data (8, 20, 23, 24) (fig. S1A). On the other hand, transcriptionally repressed genes, such as KCNA5 (25), were enriched for H3K27me3. We next compared the levels of H3K27me3 and H3K4me3 at the same loci in HUVECs and in hpMSCs. In an apparently reversed situation to the A673 EwS cell line, analysis of those promoters presented strong enrichment for H3K27me3 but not for H3K4me3 (fig. S1B). Accordingly, infection of HUVECs with the EWSR1-FLI1 oncogene (Fig. 1A) not only led to the activation of these targets (FCGRT, NR0B1, CACNB2, EZH2, IGF1, NKX2-2, and HOXD11) (Fig. 1B) but also decreased the levels of H3K27me3 (Fig. 1C). This demonstrates that, although H3K27me3 is not present at oncogene binding regions in EwS cell lines such as A673, these regions are repressed by PcG before oncogene expression.

(A) Western blot showing ectopic expression of EWSR1-FLI1 upon infection of HUVECs with an empty pLIV vector or EWSR1-FLI1pLIV. (B) RT-qPCR determination of relative mRNA expression of EWSR1-FLI1 target genes upon infection of HUVECs with an empty pLIV vector or EWSR1-FLI1pLIV. Values are normalized to TBP. (C) H3K27me3 ChIP-qPCR at EWSR1-FLI1 target gene promoters in HUVECs infected with an empty pLIV vector or EWSR1-FLI1pLIV. The values of the Y axis represent the enrichment ratio of immunoprecipitated samples relative to input with subtracted immunoglobulin G (IgG). (D) Bar plots of chromatin state relative frequencies in the whole genome [background (BG)] and in published EWSR1-FLI1 binding sites (FLI1) for three selected cell lines. Genome segmentations were extracted from the Epigenome Roadmap Consortium. (E) Heatmap with percentages of each chromatin state in the whole genome (BG) as compared to the frequency within published EWSR1-FLI1 binding regions for indicated cell lines by grouping in 8 similar chromatin states the initial classification containing 15 (quiescent segments were excluded). Bold format indicates enrichments greater than 10%. Enrichment scores were calculated as the difference between the value in EWSR1-FLI1 and the value at the whole genome, normalized by the value at the whole genome. (F) Cell proliferation expressed as cell number in 293T, A673, SK-ES1, and A4573 cells transiently transfected with small interfering RNA (siRNA) against a control (siCTRL) or two different RING1B sequences (siRING1B#1 and #2). Error bars in (B), (C), and (F) indicate SD of three biological independent experiments. Statistical significance in (D) and (F) is as follows: ***P < 0.001 and *P < 0.05.

To explore the chromatin and transcriptional states of EWSR1-FLI1 binding sites (10), we measured the frequency of each chromatin state at these regions (26) and compared to the corresponding value obtained for the whole genome in several cell lines [HUVECs, H1, and H9 human embryonic cells, H1-derived MSCs, bone marrow (BM)derived MSCs, and adipose-derived MSCs]. This analysis indicated that EWSR1-FLI1 binding sites are overrepresented in chromatin states associated with zinc finger genes and repeats (ZNF/repeats) and active promoters (Fig. 1D and table S1). In cells with MSC origin (such as H1, adipose, and BM-derived cell lines), EWSR1-FLI1 binding sites are overrepresented in PcG weak repressed state, which represents flanking regions of H3K27me3 peaks summit (Fig. 1D and fig. S1, C and D). Similar results were obtained when we grouped chromatin states of similar categories (Fig. 1E). This suggests that EWSR1-FLI1 occupies flanking regions of H3K27me3 summit peaks in hMSC, which are considered to be the potential cell of origin for EwS.

Data from our group have revealed that the PRC1 subunit RING1B, is highly overexpressed in EwS primary tumors (18). We thus assessed whether RING1B modulates the growth rate of EwS cells as has been reported for other PcG subunits, such as EZH2 and BMI1 (16, 17). RING1B depletion caused a reduction in cell viability in the A673, SK-ES1, and, with a lesser extent, in A4573 EwS cell lines but not in the control cell line 293T (Fig. 1F and fig. S1E), suggesting that RING1B represents an epigenetic vulnerability for EwS cells.

Chan et al. (27) recently proposed that RING1B might play a role in modulating enhancer activity. Together with its role in promoter regulation, EWSR1-FLI1 has been recently reported to generate de novo enhancers (10). This led us to postulate whether EWSR1-FLI1 and RING1B might cooperate during EwS tumorigenesis. We first aimed to define the genome-wide localization of RING1B and its repressive histone mark H2Aub in the A673 cell line by chromatin immunoprecipitation sequencing (ChIP-seq). In two independent experiments, we identified 2573 and 3945 peaks of RING1B, and 26424 and 10269 peaks of H2Aub. Using differential binding analysis (DiffBind), which allows for the identification of statistically common peaks (28), we found 2459 RING1B and 5392 H2Aub significant peaks between duplicates (P < 0.05, fig. S2A), corresponding to 1264 target genes and 3013 target genes, respectively (table S2). Genomic distribution of peaks showed that RING1B is more abundant in intergenic regions, whereas H2Aub is mainly located in promoters (Fig. 2A). Moreover, 38% of RING1B peaks were found at intergenic regions with respect to 21.5% of H2Aub peaks, and 29.2% of RING1B peaks were in promoters with respect to 40.5% of H2Aub peaks, further supporting the potential role of RING1B at enhancers. We then categorized peaks for RING1B, H2Aub, and EWSR1-FLI1 in active or poised enhancers, and in active or poised promoters, based on H3K27me3, H3K4me3, H3K27ac, and H3K4me1 (29). To complement the above data, we performed a ChIP-seq analysis using a different antibody directed against FLI1 (fig. S2B and table S2). We found that an important fraction of RING1B peaks (35%) and EWSR1-FLI1 (46%) are located at transcriptionally active enhancers and promoters of A673 cells (Fig. 2B, left). On the other hand, as expected, 35% of RING1B peaks and 37% of H2Aub peaks showed a preference for transcriptionally repressed regulatory regions (Fig. 2B, left). We then intersected the list of genes associated to RING1B and H2Aub peaks with published data of EWSR1-FLI1 target genes in A673 cells, producing a common set of 162 genes (fig. S2C and table S3). Comparing this set with 386 genes containing only RING1B and H2Aub or the group of 324 EWSR1-FLI1/RING1B genes without H2Aub confirmed that the presence of EWSR1-FLI1 correlated with higher level of transcription (P < 1016; fig. S2D, left). Functional analysis of the common gene set of 324 EWSR1-FLI1/RING1B genes (table S3) returned Gene Ontology (GO) categories related to chondrocyte and neuronal differentiation (fig. S2D, right). EWSR1-FLI1/RING1B/H2Aub genes were also enriched in neuronal differentiation category, while the RING1B/H2Aub genes were related to general transcription. These data suggest that RING1B is a positive regulator of a specific set of genes implicated in EwS and that this activity is independent of its canonical repressive mark.

(A) Pie chart showing genomic distribution of RING1B and H2Aub peaks relative to functional categories including promoter (2.5 kb from TSS), gene body (intragenic region not overlapping with promoter), and intergenic (rest of the genome). (B) Boxplot depicting percentage of regulatory elements (active/bivalent enhancers and promoters) in each described group. (C) Venn diagram depicting the overlap between RING1B and EWSR1-FLI1 in A673 cells at the peak level. (D) Aggregated plot showing the average ChIP-seq signal of RING1B and EWSR1-FLI1 at EWSR1-FLI1 binding sites. (E) Aggregated plots showing the average ChIP-seq signal of H3K27ac, H2Aub, and H3K27me3 in the three sets of RING1B and EWSR1-FLI1 peaks. (F) Heatmap showing RING1B, EWSR1-FLI1, H3K27ac, H2Aub, and H3K27me3 ChIP-seq signals segregating in the three sets of RING1B and EWSR1-FLI1 peaks. Top MEME motif for every group is shown. (G) University of California Santa Cruz (UCSC) genome browser ChIP-seq signal tracks for RING1B, EWSR1-FLI1, H2Aub, H3K27ac, H3K4me3, and H3K27me3 at NKX2-2, CCND1, VRK1, and CAV1 gene promoters and intergenic enhancer regions. Gray boxes represent EWSR1-FLI1 and RING1B colocalization and ES super-enhancers (SEnh; as shown at VRK1 and CAV1/2).

To fully understand the association of RING1B with transcriptional activation in EwS, we intersected EWSR1-FLI1 peaks with those of RING1B and obtained 955 common regions (Fig. 2C). Notably, intersection between H2Aub and RING1B peaks returned only 589 common peaks. Among the 955 overlapping EWSR1-FLI1/RING1B peaks, we inspected for genes containing an enhancer within 100 kb and obtained 1276 genes, of which 235 (18%) were reported to be regulated by EwS super-enhancers (table S4) (11). The common targets of RING1B and EWSR1-FLI1 sites were found within active enhancers, while the majority of RING1B peaks not overlapping with EWSR1-FLI1 were located in transcriptionally repressed regulatory elements (Fig. 2B, right). The distribution of RING1B peaks was centered on EWSR1-FLI1 binding sites (Fig. 2D), suggesting that their binding occurs at the same loci. We next assessed the distribution of H3K27ac, H2Aub, and H3K27me3 in genomic regions occupied by EWSR1-FLI1, RING1B, or shared (Fig. 2E). Common peaks were decorated with H3K27ac, lacking H2Aub (Fig. 2, E and F), and presented narrow RING1B peaks located in intergenic or intronic regions (fig. S2E, right). These data suggest that common sites likely represent enhancers. Known EWSR1-FLI1 target genes such as NKX2-2, CCND1, VRK1, or CAV1 presented an intergenic peak of RING1B, which overlaps with defined super-enhancers in the case of VRK1 and CAV1 (Fig. 2G). Intronic enhancers such as JARID2 or MYOM2 (fig. S2G) constitute the majority of the 162 common RING1B, EWSR1-FLI1, and H2Aub genes (53% of sites, fig. S2C). On the other hand, RING1B-specific peaks were associated with H3K27me3 and H2Aub (Fig. 2, E and F) and presented a broader distribution [e.g., HNF1B and TAL1 (fig. S2H)] mainly located within promoter or gene body regions (fig. S2E, left). The bivalent marks H3K4me3 and H3K27me3 decorated 63% of the 932 downstream genes associated to RING1B-specific peaks (P < 10300, table S4) (29). RING1Btranscription start sites (TSS) do not overlap with EWSR1-FLI1 and are decorated with H2K27me3 and H2Aub, while RING1B-distal sites overlap with EWSR1-FLI1 and with H3K27ac (fig. S2F).

Last, de novo motif analysis revealed that EWSR1-FLI1specific sites contained predominantly (P < 10282) one single occurrence of the canonical ETS motif GGAA (Fig. 2F). When EWSR1-FLI1 was associated with RING1B, we observed a significant enrichment for multimeric GGAA repeats (P < 101072) (10). Furthermore, RING1B-sepecific sites were enriched for CG sequence, as previously reported (P < 10176) (30). Together, we identified two major types of RING1B peaks in EwS: a prominent group with narrow peaks that colocalizes with EWSR1-FLI1 at enhancers of actively transcribed genes and a second group with broader peaks located at promoters, where RING1B is associated with H2Aub.

To further characterize RING1B binding regions (table S4), we analyzed several EWSR1-FLI1 active promoters (CAV1, FCGRT, NR0B1, CACNB2, FEZF1, and KIAA1797) and enhancers (CCND1, IGF1, CAV2, JARID2, VRK1, and NKX2-2) by ChIP-qPCR. Both groups showed enrichment for RING1B, with stronger signals at enhancers (Fig. 3A). Known repressed targets of the oncogene (e.g., IGFBP3, TGFBR2, and LOX) also showed binding of RING1B. At these repressed promoters, RING1B was accompanied by its canonical repressive mark H2Aub (fig. S3A). We also validated the occupancy of RING1B in EWSR1-FLI1activated promoters (CAV1, FCGRT, NR0B1, and FEZF1) and enhancers (CCND1, CAV2, JARID2, and VRK1) in SK-ES1 cells (fig. S3B). Similar to A673 cells, H2Aub correlated with RING1B at promoters of repressed genes (IGFBP3, TGFBR2, and LOX) (fig. S3C). Last, we observed that the PRC1 and PRC2 subunits, BMI1 and EZH2, respectively, were present at repressed promoters but not in active enhancers (fig. S3, D and E), as well as in promoters with broad peaks of RING1B concomitant with H3K27me3 and H2Aub but no EWSR1-FLI1 (e.g., TAL1, IGF1R, and HNF1B) (fig. S3F). Furthermore, genome-wide analysis demonstrated that BMI1 and CBX7 (31) subunits of the PRC1 canonical complex colocalize with RING1B only at repressed regions (TAL1) as shown in Fig. 3B, while no detectable peaks are present at active enhancers where EWSR1-FLI1 is present (VRK1). Thus, while RING1B decorates EWSR1-FLI1activated promoters and enhancers, it also maintains its canonical role at several oncogene repressed regions, as well as in a subgroup of genes with no EWSR1-FLI1.

(A) RING1B ChIP-qPCR of EWSR1-FLI1 bound active promoters, repressed promoters, and active enhancers. Control regions indicate the absence of RING1B and EWSR1-FLI1 binding at these sites. The values of the Y axis represent the enrichment ratio of immunoprecipitated samples relative to input. (B) UCSC genome browser ChIP-seq signal tracks for EWSR1-FLI1, RING1B, CBX7, BMI1, H2Aub, and H3K27me3 at TAL1 promoter and VRK1 enhancer. (C) Histogram depicting percentages of activated and repressed genes in A673 and SK-ES1 cells with stable RING1B knockdown seq#2 (shRING1B#2) versus control seq#2 (shCTRL#2), with P < 0.05 and an absolute fold change (FC) > 1.25 or 1.5. (D) Western blot showing RING1B, RING1A, H2Aub, and H3K27me3 in A673 and SK-ES1 cells with either shCTRL#2 or shRING1B#2. Lamin B and histone H4 are used as loading controls. (E) Venn diagram showing intersection between differentially activated or repressed genes for EWSR1-FLI1 and RING1B in A673 cells; P < 0.05. (F) RT-qPCR determination of mRNA expression of EWSR1-FLI1 target genes with active enhancers in shCTRL and shRING1B A673 cells (#1 and #2). Values are normalized to GAPDH. (G) Same analysis as in (F) for SK-ES1 cells. Error bars in (A), (F), and (G) indicate SD of four independent biological experiments and ***P < 0.001, **P < 0.01, and *P < 0.05.

To understand whether RING1B behaves as a canonical repressor and/or activator in EwS, we analyzed the expression changes after knocking down RING1B using two different sets of short hairpin RNA (shRNA, seq#1 and seq#2; fig. S4A). The data obtained showed that 71.94 and 63.85% of genes were down-regulated in the A673 and SK-ES1 cell lines, respectively (FC < -1.5, Fig. 3C). This confirms our finding that RING1B acts predominantly as an activator, despite its presence at several EWSR1-FLI1repressed targets. Furthermore, H2Aub levels remained unchanged after RING1B knockdown (Fig. 3D), while RING1A knockdown produces a notable decrease in H2Aub levels (fig. S4B). These data suggest that RING1B main function in EwS is uncoupled from its ubiquitin ligase activity toward H2A and that RING1A is the main histone H2A mono-ubiquitin ligase. To further elucidate to what extent RING1B cooperates with EWSR1-FLI1 in transcription regulation, we intersected differentially expressed genes in RING1B knockdown cells (absolute FC > 1.25) with those affected by EWSR1-FLI1 knockdown (absolute FC > 1.5) (10), obtaining an overlap of 1078 genes. After segregating these data into down- and up-regulated genes, we found that RING1B and EWSR1-FLI1activated 229 genes and repressed 162 genes (Fig. 3E and table S5). Among the 229 activated genes, we found several developmental genes, including SOX2, SIX3, LYAR, and KIT. GO analysis showed regulation of the potassium channel and mechanisms that control actin monomers and filaments as the main categories (fig. S4C), in agreement with previous publications (25, 32). Among the activated genes, SOX2 and KIT harbored RING1B and EWSR1-FLI1 peaks in intergenic and intronic enhancer regions, respectively (fig. S4E). TGFBR2, a gene repressed by both EWSR1-FLI1 and RING1B, also contained an intronic enhancer where both proteins colocalized. Notably, the expression of known targets of EWSR1-FLI1, such as NKX2-2 or IGF1 (fig. S4D), was just below our logFC cutoff value. Nonetheless, we confirmed by reverse transcription (RT)qPCR the changes in expression levels of selected repressed and activated genes cobound by EWSR1-FLI1 and RING1B. We noticed that RING1B knockdown causes a significant reduction in the expression levels of those genes where both EWSR1-FLI1 and RING1B were co-occupying enhancer regions (Fig. 3, F and G). The expression of CAV1, NKX2-2, SOX2, IGF1, JARID2, and VRK1 was affected in stronger manner upon EWSR1-FLI1 knockdown, indicating that some cofactors could remain when RING1B is depleted (fig. S4F). The effect of RING1B knockdown was less pronounced when both proteins were enriched at promoter regions of active genes (fig. S4, G and H, left). As expected, at those genes where EWSR1-FLI1 acts as a repressor, RING1B knockdown induces a promoter reactivation (fig. S4, G and H, right). Overall, these data indicate that RING1B and EWSR1-FLI1 cooperate in gene activation, at both the promoter and enhancer levels, while RING1B retains its canonical role at those targets repressed by the oncogene. Since a large number of EWSR1-FLI1 and RING1B cotargets were not altered by RING1B knockdown, we postulate compensatory mechanism(s) or additional cofactors involved in their regulation.

Wild-type EWSR1 interacts with RING1B in the VCaP prostate cancer cell line (33). We also confirmed this interaction in SK-ES1 cells (Fig. 4A). Since RING1B and EWSR1-FLI1 are enriched at transcriptionally active regions, we next aimed to investigate whether both proteins interact. Coimmunoprecipitation experiments in HeLa cells where EWSR1-FLI13xFlag was overexpressed (34) confirmed that indeed oncogene interacts with RING1B (Fig. 4, B and C). Analysis of published mass spectrometry data demonstrated that several SWI/SNF subunits interact with RING1B (33), further supporting an active role of RING1B in EwS gene regulation. Together, our results indicate that EWSR1-FLI1 and RING1B not only colocalize at the same genomic regions but also physically interact, mainly through the EWSR1 component of the fusion protein.

(A) Western blot showing endogenous coimmunoprecipitation of RING1B with EWSR1 in the SK-ES1 cell line. (B) Western blot showing overexpression of EWSR1-FLI1-3xFlag and RING1B levels in HeLa stably transfected cells upon induction with indicated doxycycline concentrations for 24 hours. Calnexin is used as loading control. (C) Coimmunoprecipitation of RING1B with EWSR1-FLI1-3xFlag under induction conditions (0.5 g/ml). Inputs in (A) and (C) contain 10% of immunoprecipitated material and IgG is used as control. (D) Western blot showing RING1B and EWSR1-FLI1 in cytoplasm, soluble, and bound chromatin fractions in shCTRL#1 or shRING1B#1 SK-ES1 cells. Histone H4 is used as a control of bound chromatin, and GAPDH as a control of cytoplasmic fraction. Blot quantification of the same ordered samples is depicted below. (E) ChIP-qPCR analysis of FLI1, RING1B, and H3K27ac at EWSR1-FLI1activated enhancers of NKX2-2, SOX2, or IGF1 genes in shCTRL#2 and shRING1B#2 A673 cells. ENC1 is used as negative control region. The values of the Y axis represent the enrichment ratio of immunoprecipitated samples relative to input. Error bars indicate SD of three independent biological experiments. Statistical significance is as follows ***P < 0.001, **P < 0.01, and *P < 0.05. (F) Aggregated plot and boxplot showing the average ChIP-seq signal of RING1B and FLI1 peaks at RING1B and EWSR1-FLI1 binding sites, respectively, in shCTRL#2 and shRING1B#2 A673 cells. (G) UCSC genome browser ChIP-seq signal tracks for EWSR1-FLI1 and RING1B in shCTRL#2 and shRING1B#2 A673 cells at SOX2 and VRK1 enhancer regions.

Next, we analyzed whether RING1B depletion affects the EWSR1-FLI1 recruitment to chromatin. As expected, after knockdown, we observed a notable reduction of RING1B in the chromatin bound fraction (Fig. 4D). EWSR1-FLI1 was also evicted from chromatin bound and enriched in the soluble chromatin fraction (Fig. 4D). We then monitored the occupancy of EWSR1-FLI1, RING1B, and H3K27ac at several enhancers (e.g., SOX2, NKX2-2, and IGF1). The data in Fig. 4E showed that upon RING1B knockdown, enrichments at those enhancers decreased to control values [immunoglobulin G (IgG) or ENC1 region]. To assess the decrease of EWSR1-FLI1 recruitment genome-wide, we performed ChIP-seq analysis of RING1B and FLI1 in shCTRL and shRING1B A673 cells. The analysis indicated that upon RING1B depletion, EWSR1-FLI1 binding to chromatin was reduced (Fig. 4, F and G). In sum, we conclude that in EwS, RING1B exerts its main role as activator by promoting recruitment of EWSR1-FLI1 to enhancer regions.

RING1B stimulates tumor growth and metastasis in melanoma, leukemia, and breast cancers (14, 27). We observed a reduction in colony number when RING1B is depleted in the SK-ES1 cell line (fig. S5A). To gain functional insight into the cancer pathways potentially modulated by RING1B, we performed gene set enrichment analysis (GSEA) by comparing SK-ES1 shCTRL versus shRING1B cells. The top 10 most significant pathways included interferon-, epithelial-to-mesenchymal transition, hedgehog signaling, and angiogenesis, with a 0.25 Q value cutoff (fig. S5B). In EwS, disruption of angiogenic pathways has been described (4, 22). Further inspection of angiogenic gene list revealed that key genes such as PDGFA, FGFR1, SLCO2A1, CXCL6, and S100A4 were down-regulated upon RING1B depletion (fig. S5C).

To assess the relevance of RING1B in vivo, we generated xenografts by injecting SK-ES1 shCTRL or shRING1B cells (seq#1 and seq#2) subcutaneously into athymic nude mice. Cells with reduced RING1B levels showed delayed engraftment and slower tumor growth (Fig. 5A). At 21 days after injection, tumors derived from shRING1B cells were significantly smaller than those from control cells (fig. S5D). Notably, the median survival increases from 26 days for shCTRL cells to 30 days for shRING1B seq#1 and from 20 to 27 days for shRING1B seq#2 (Fig. 5B). Immunohistochemical analyses of tumors confirmed reduced levels of RING1B, while the ES marker CD99 remained essentially unchanged (Fig. 5C and fig. S5E). Furthermore, shCTRL tumors displayed higher proliferation rates than shRING1B, as shown by Ki-67 staining (Fig. 5C).

(A) Tumor volume curve in xenografts established by subcutaneous injection of shCTRL and shRING1B#1 (n = 9 and n = 10, respectively, above) or shRING1B#2 (n = 12 both groups, below) SK-ES1 cells in athymic nude mice. (B) Kaplan-Meier xenograft survival curves in shCTRL and shRING1B SK-ES1 cells (#1 and #2). (C) Immunohistochemistry staining of EWSR1-FLI1, CD99, and RING1B on sections of tumors excised from shCTRL#1 and shRING1B#1 SK-ES1 xenografts. Proliferation was analyzed by Ki67 immunohistochemistry; hematoxylin and eosin (H&E) was used as control. (D) Heatmap depicting fold changes in gene expression in six tumors excised from shCTRL#1 and shRING1B#1 SK-ES1 groups. (E) RT-qPCR levels of mRNA expression for RING1B and EWSR1-FLI1 in shCTRL#1 and shRING1B#1 SK-ES1derived tumors; ***P < 0.001. (F) RT-qPCR levels of mRNA expression for genes regulated by EWSR1-FLI1/RING1B enhancers (left) and angiogenic genes (right) in shCTRL#1 and shRING1B#1 SK-ES1 derived tumors; *P < 0.05.

To better characterize xenograft derived tumors, we performed RNA sequencing (RNA-seq) of a cohort of tumors (six for each group, Fig. 5D). GSEA analysis confirmed the enrichment of angiogenic genes in the shCTRL tumors (fig. S5, F and G). Since RING1B retains its repressive function at several promoters, we hypothesized that the delay in survival and in tumor growth upon RING1B knockdown could be related to up-regulation of tumor suppressor genes (TSG). GSEA applied to 983 genes from TSG database (https://bioinfo.uth.edu/TSGene), indicated that this gene list was enriched in shCTRL phenotype, suggesting that tumor growth and survival differences observed were not due to RING1B repression of TSG (fig. S5H). The NKX2-2, SOX2, and IGF1 genes are necessary for EwS tumor proliferation (21, 23, 35). In agreement, confirmed RING1B and EWSR1-FLI1 expression reduction (Fig. 5E) is associated to down-regulation of these genes in xenograft tumors (Fig. 5F, left), as we previously shown in EwS cells (Fig. 3, F and G). Furthermore, after RING1B knockdown, we also validated down-regulation of S100A4, SLCO2A1, and VEGFA, which are main activators of angiogenic signaling pathways (Fig. 5F, right). All these data highlight the role of RING1B as an activator in EwS tumorigenesis.

Several kinases (including AURKB, MEK1, and CK2) have been reported to modulate the activating transcriptional function of RING1B (14, 15, 36). To investigate which pathway(s) regulates RING1B at active enhancers in EwS, we analyzed the expression levels of these three kinases in a publicly available database (4) comprising a cohort of 27 tumor samples and BM-MSCs. While MEK1 and CK2 were not expressed in primary tumors with respect to BM-MSCs (control), 11 of 27 EwS tumors (40%) showed higher levels of AURKB compared to control (fig. S6A). EWSR1-FLI1 directly regulates the expression of AURKB (37), as also demonstrated by AURKB down-regulation in EwS cell lines upon oncogene knockdown (fig. S6A).

AZD1152 is a specific AURKB inhibitor, with a median inhibitory concentration (IC50) of 19 nM in EwS cell lines (38). Accordingly, we observed IC50 values of 5 and 6 nM in SK-ES1 and A4573 cells, respectively; in contrast, the IC50 for A673 was 5 M, and AZD1152 had no effect on the control cell line 293T (fig. S6B). EwS cells that survived to the treatment showed an atypical phenotype, suggesting enhanced differentiation (fig. S6C). Furthermore, viability of EwS cell lines was not affected by the inhibition of RING1B E3 ubiquitin ligase activity with PRT4165 (fig. S6B). To further elucidate the effect of AZD1152 in EwS, cell death was analyzed by Annexin V staining. A 72-hour AZD1152 treatment of A673, SK-ES1, and A4573 cells led to an increase in the early and late apoptosis populations as compared to 293T cells (Fig. 6A). Analysis of cleaved PARP levels further demonstrated that AZD1152 stimulated apoptotic pathways in EwS cell lines, with SK-ES1 being the most sensitive (Fig. 6B). It is worth noting that the levels of EWSR1-FLI1 were decreased after AZD1152 treatment in SK-ES1 and A4573, yet RING1B levels were unaffected (Fig. 6B and fig. S6, D and E, right). To understand how AURKB modulates RING1B in EwS, we analyzed H2Aub levels after AZD1152 treatment. We observed increased levels of H2Aub repressive mark after AURKB inhibition, suggesting that this kinase indeed inhibits the ubiquitin ligase activity of RING1B in EwS (Fig. 6C). Furthermore, in SK-ES1 and A4573 cells, the increase in ubiquitin ligase activity correlated with decreased expression of EWSR1-FLI1 targets co-occupied by RING1B, with more pronounced effect on those genes where both proteins colocalize at the enhancer region (Fig. 6D and fig. S6, D and E, left). For the A673 cell line, higher doses were required to reach oncogene target deregulation, as expected. Next, we reasoned that AURKB should be present at those regions where it inhibits RING1B activity. Using ChIP-qPCR, we demonstrated that AURKB is enriched in active enhancers (CAV2, driving CAV1 expression, and SOX2; Fig. 6E) and promoters (NR0B1; fig. S6F). Furthermore, EWSR1-FLI1 down-regulation could be explained by the presence of RING1B at the EWSR1 promoter, which indirectly decreases upon AZD1152 incubation (fig. S6G). Although part of AZD1152 cytotoxicity might be related to reduction of EWSR1-FLI1 availability, the data presented suggest that RING1B regulation of oncogene targets is susceptible to AURKB inhibition. The translational value of this potential targetable vulnerability is the matter of ongoing work.

(A) Annexin V staining of SK-ES1, A673, and A4573 cells after treatment with AZD1152 (20 nM). 293T cells were used as a control cell line. (B) Western blot analysis of cleaved poly(ADP-ribose) polymerase (cPARP), EWSR1-FLI1, RING1B, and AURKB after treatment with 10 or 20 nM AZD1152, in the A673, SK-ES1, A4573, and 293T cell lines. Tubulin was used as loading control. (C) Western blot analysis of H2Aub and H3S10phospho (H3S10ph) in the A673, SK-ES1, and A4573 cell lines treated with 5 or 20 nM AZD1152. Histone H4 was used as loading control. (D) RT-qPCR determination of mRNA expression of target genes with RING1B/EWSR1-FLI bound enhancers in SK-ES1 and A4573 cells after treatment with 20 nM AZD1152. RPL27 was used for normalization. DMSO, dimethyl sulfoxide. (E) AURKB ChIP-qPCR at CAV2 and SOX2 EWSR1-FLI1/RING1B enhancers (above) and control regions (below). The values of the Y axis represent the enrichment ratio of immunoprecipitated samples relative to input. Error bars in (D) and (E) indicate SD of three independent biological experiments. (F) Schematic representation illustrating the EWSR1-FLI1 recruitment by RING1B to repressed regions containing GGAA repeats. Once EWSR1-FLI1 has been recruited, additional cooperating factors such as AURKB might inhibit RING1B ubiquitin ligase activity, which, in turn, is able to participate in transcription activation.

Here, we investigated the genome-wide occupancy of RING1B in EwS. In agreement with previous data, we identified a set of regions bound by RING1B where it exerts its canonical repressive function. We also report that RING1B co-occupy together with EWSR1-FLI1 many intergenic and intronic regions decorated with H3K27ac. A strong enrichment in GGAA repeats has been described in regulatory elements where EWSR1-FLI1 binds producing active enhancers (10). The presence of GGAA repeats, as well as the H3K27ac association, indicates that cobinding of RING1B and EWSR1-FLI1 occurs in active enhancers. BMI1 or EZH2 was not found at these enhancer regions, suggesting a Polycomb-independent function for RING1B. Enhancers are key regulatory regions implicated in cell fate determination. Here, we unveiled that an aberrant transcription factor such as EWSR1-FLI1 relies on RING1B to activate enhancers, causing an altered gene expression profile, which favor cell transformation.

In accordance with RNA-seq data from melanoma and breast cancer, where a positive association of RING1B with transcription activation has been reported (14, 27), we observed in EwS cells a higher number of genes activated than repressed by RING1B. We found NKX2-2, SOX2, and IGF1 being direct targets down-regulated both in vivo and in vitro upon RING1B knockdown. In EwS, NKX2-2 and SOX2 are key players in tumorigenesis (21, 23), suggesting that modulation of their expression in vivo upon RING1B knockdown might contribute to decreased tumor volume and better survival, supporting an oncogenic role for RING1B.

Recent studies in hpMSCs have demonstrated that, before oncogene recruitment, H3K27me3 is enriched at regions where EWSR1-FLI1 could bind (39). In agreement with these data, we further demonstrate that upon EWSR1-FLI1 expression, those same regions loose H3K27me3 marks while becoming transcribed. Moreover, we report that enrichment in Polycomb repressed chromatin states is specific for H1-, adipose- and BM-derived MSCs, reinforcing hMSC as the putative cell of origin, which has already been described by other groups (4, 21). The existence of H3K27me3 repressed regions decorated only with PRC1 complex has already been described during differentiation of neural precursor cells, where RING1B and PCGF2 are retained while the PRC2 subunit Suz12 is not (40). In melanoma, CCND2 is marked with H3K27me3 before RING1B activation by phosphorylation (14). We have observed that GGAA repeats are differentially enriched in the binding motif analysis when RING1B is associated to chromatin with EWSR1-FLI1. In this scenario, given the interaction observed for RING1B and EWSR1-FLI1, it is tempting to speculate that RING1B targets EWSR1-FLI1 to specific sites. In line with this hypothesis, the reduced recruitment of EWSR1-FLI1 to chromatin (including enhancer regions, such as NKX2-2, SOX2, and IGF1) upon RING1B knockdown underlines the importance of RING1B in the initials steps of EwS tumorigenesis. Overall, our data suggest that RING1B is required for the recruitment of EWSR1-FLI1 to multimeric GGAA repeats (Fig. 6F).

We have demonstrated that RING1B is an essential partner of EWSR1-FLI1 triggering chromatin remodeling. Recent studies demonstrated the requirement of SWI/SNF, WDR5, and p300 acetyltransferase for EWSR1-FLI1induced transcription. Similarly, in synovial sarcoma, the SS18-SSX oncogenic fusion protein and the SWI/SNF complex colocalize at KDM2B-repressed target genes together with the noncanonical PRC1.1 complex to produce transcriptional active regions (41). Along the same lines, in leukemia, noncanonical PRC1.1 also targets active genes independently of H3K27me3 (42). Further mechanistic insights are needed to elucidate the contribution of PRC1.1 repressive complex in EwS, where somatic mutations in BCOR have been reported (1). The noncanonical PRC1.1 complex contains a DNA binding ZnF-CXXC domain able to target chromatin via KDM2B (43). ZNF/repeats chromatin state was statistically enriched in five of the six EwS cell lines analyzed.

Recently, different cell models have shown that the E3 ubiquitin ligase activity of RING1B is inactivated by phosphorylation (15, 36). Our results showing the recruitment of AURKB to enhancers are compatible with a model in which RING1B is unable to repress the newly formed ES enhancers, which were previously Polycomb-repressed regions. Once the oncogene binds to chromatin, RING1B would cooperate to induce transcription activation if its ubiquitin ligase activity is inhibited by phosphorylation (either directly or indirectly) (Fig. 6F). More studies are needed to clarify how oncogenic fusion proteins act as binding scaffolds to recruit a specific set of interactors to generate previously unknown functional units (such as neo-enhancers).

Inhibition of super-enhancers activity with BET inhibitors has emerged as a successful preclinical strategy in the fight against different pediatric cancers such as EwS, neuroblastoma, and rhabdomyosarcoma (4446). Inhibition of AURKB with AZD1152 increases H2Aub and decreases expression of key oncogene targets, thus suggesting that RING1B is essential for enhancer deregulation by EWSR1-FLI1. Nevertheless, as RING1B account for catalytic and noncatalytic dependencies (14), further investigation should address its clinical therapeutic implications. In agreement with our data, combined inhibition of AURKA and AURKB, as well as synergistic activity of AURKB with focal adhesion kinase inhibitors, has been described effective in EwS preclinical studies, although AURKB efficiency as single agent has not been proved (47, 48). In EwS cells, AZD1152 could affect the levels of RING1B, and this likely reverberates on the regulation of the oncogenes promoter since RING1B occupies the EWSR1 promoter (fig. S6, E and G).

In summary, we demonstrate the oncogenic dependency to high levels of RING1B in EwS. The data support a model in which RING1B plays a pivotal role for EWSR1-FLI1 recruitment to the multimeric DNA repeats. This, in turn, allows for transcriptional activation that defines the characteristic transcriptome of EwS. Given the role of RING1B in the activation of super-enhancers, which are critical elements for cell fate determination, we propose that the EwS cell of origin is predefined by high levels of RING1B.

The Ewings sarcoma cell lines A673, SK-ES1, and A4573, which carry the EWSR1-FLI1 translocation types I, II, and III, respectively, and the HEK293 cell line from human embryonic kidney infected with AgT from SV40 (293T), were cultured in RPMI 1640 media (Gibco) and supplemented with 10% fetal bovine serum, l-glutamine, and penicillin/streptomycin. Cells were cultured at 37C with 5% CO2. The A673 and SK-ES1 cell lines harboring shCTRL and shRING1B with seq#1 and seq#2 as well as A673 cell line with doxycycline inducible knockdown of EWSR1-FLI1 were previously described (11, 18). hpMSCs were isolated following published protocols (21). Ectopic expression of EWSR1-FLI1 3xFLAG C terminus in HeLa cells was induced with doxycycline (0.5 g/ml) (34).

All experiments performed with AZD1152 were incubated 72 hours, with the exception of RNA expression assays that were incubated 24 hours. For IC50 calculations, A673, SK-ES1, A4573, and 293T cell lines were seeded at 2000 cells per well in 96-well culture plates. AZD1152 and PRT4165 (Sigma-Aldrich) was added to complete growth medium; after 72 hours, cells were subjected to the ATPlite assay (PerkinElmer), and measurements were performed using a Tecan plate reader. Inhibitory concentrations were calculated using OriginPro 9.0 software.

EWSR1-FLI1 type 2 was amplified from a pSG5 vector with primers containing Bgl II and Hind III sequences (forward, 5-ggaggaaggAGATCTAATGGCGTCCACGG-3; reverse, 5-aagAAGCTTGTAGTAGCTGCCTAA-3). The PCR product was purified using an Illustra GFX PCR DNA and Gel Band Purification kit (GE Healthcare Life Sciences). The product of the amplification was subcloned into the TOPO TA Cloning Kit for Sequencing following the manufacturers instructions. TOPO-EWSR1-FLI1 plasmid and the acceptor vector pEGFP-N1 were double digested with Bgl II and Hind III at 37C. The resulting EWSR1-FLI1 band was ligated into pEGFP-N1, and ligation product was then transformed into JM109 cells.

Target sequences for siRNA are described in table S6. Transfection of small duplexes (Sigma-Aldrich) was performed with Lipofectamine RNAiMAX and Optimem (Invitrogen), using 30 pmol when cells were 80% confluent; samples were collected after a 72-hour incubation. Transient transfections of GFP constructs or empty vector were done using FuGENE XP (Roche) with 1 to 2 g of plasmid when cells were 60% confluent; samples were collected after 48 hours. Both reagents were used according to the manufacturers recommendations.

Empty pLIV and EWSR1-FLI1pLIVexpressing lentiviruses were provided by N. Riggi (University Institute of Pathology Lausanne, Switzerland). Lentiviruses were produced in Lenti-X 293T packaging cells (Takara, Cultek) at a low passage number. For each plate, 7 g of the lentiviral plasmid, 5 g of the envelope plasmid (VSV-G), and 6 g of the packaging plasmid (PAX8) were prepared and introduced by calcium phosphate transfection, according to standard protocols. The supernatant containing lentiviruses was collected 48 hours after transfection. The HUVEC cell line was seeded at 3000 cells/cm2 and transduced with 3:1 of the lentiviral supernatant with fresh media containing Polybrene (Sigma-Aldrich) at 6 g/ml. Cells were selected with fresh growth media containing puromycin (0.3 g/ml) for 72 hours. A control dish without the transduction media was also selected with puromycin, to control for killing of nontransduced cells.

Histone extracts of cultured cells were isolated using the EpiQuick Histone Extraction kit (Epigentek) following the manufacturers instructions. Total cell extracts were prepared in IPH buffer [50 mM tris-HCl (pH 8), 150 mM NaCl, 5 mM EDTA, and 0.5% NP-40] with EDTA-free protease inhibitor cocktail (Roche). For protein, fractionation standard protocols were used. Histone or total protein extracts were quantified by Bradford assay. Immunoprecipitation was performed with total cellular extracts incubated at 4C overnight with primary antibody. After incubation of immunoprecipitated samples on protein A/G and agarose beads (Santa Cruz Biotech), 30 to 50 g of whole protein extracts or 5 g of histones was resolved by polyacrylamide gel electrophoresis. Western blotting was performed using standard protocols. Incubation with primary antibodies was done at 4C overnight and LI-COR secondary antibodies that are detectable by near-infrared fluorescence were used for detection (table S6). Blots were scanned with an Odyssey CLx Infrared Imaging System at medium intensities.

Treated cells were fixed in 70% ethanol, stained with 25 l of propidium iodide (PI) (1 mg/ml), and 25 l of ribonuclease (RNase) (10 mg/ml), and incubated 30 min at 37C. For Annexin V binding, the Alexa Fluor 488 fluorophore kit (Invitrogen) was used for apoptotic cell detection. After culture and treatment, cells were resuspended in annexin binding buffer with 5 l of Alexa Fluor 488 Annexin V and 1 l of PI working solution (100 g/ml). After 15 min, samples were run in Gallios multicolor flow cytometer (Beckman Coulter) set up with the 3-lasers, 10 colors standard configuration. Histograms and cytograms were further analyzed with FlowJo 10.2.

Total RNA was isolated and purified from collected cells using the RNeasy Mini Kit (Qiagen) according to the manufacturers protocol. After quantification using the NanoDrop software (Thermo Fisher Scientific), RT was performed. A 1-g aliquot of each RNA sample was converted to cDNA in a reaction catalyzed by a retrotranscriptase enzyme (M-MLV Reverse Transcriptase Promega). Random primers and RNase inhibitor (RNasin Plus RNase Inhibitor, Promega) were also added to the reaction. cDNA obtained was analyzed by qPCR using SYBR Green PCR Master Mix (ABI). cDNA was amplified with specific oligonucleotides (table S6). Each cDNA sample was run in triplicate, and its levels were analyzed using the 7500 Fast PCR instrument (Applied Biosystems). To compare between different conditions studied, relative quantification of each target was normalized to a housekeeping gene. Last, data were analyzed using the comparative 2-ct method.

Gene expression microarrays were performed at the Microarray Analysis Service, Hospital del Mar Medical Research Institute (IMIM, Barcelona). RNA samples were amplified, labeled according to a GeneChip WT PLUS Reagent kit, and hybridized to Human Gene 2.0 ST (Affymetrix) in a GeneChip Hybridization Oven 640. Washing and scanning were performed using the Expression Wash, Stain, and Scan Kit and the GeneChip System of Affymetrix (GeneChip Fluidics Station 450 and GeneChip Scanner 3000 7G). After quality control, raw data were background corrected, quantile-normalized, and summarized to a gene level using the robust multichip average; a total of 48,144 transcript clusters, excluding controls, were obtained, which roughly corresponds to genes and other RNAs, such as long intergenic noncoding RNAs and microRNAs. NetAffx 36 annotations, based on the human genome 19, were used to summarize data into transcript clusters and to annotate analyzed data. Linear Models for Microarray (limma), a moderated t statistics model, was used for detecting differentially expressed genes between the conditions. All data analyses were performed in R (version 3.4.3) with R/Bioconductor packages aroma.affymetrix, Biobase, affy, limma, genefilter, ggplots, and Vennerable. Genes with a P less than 0.05 were selected as significant.

Raw sequencing reads in the fastq files were mapped with STAR version 2.6.a (49). GENCODE release 29, based on the GRCh38 reference genome, and the corresponding GTF file were used. The table of counts was obtained with featureCounts function in the package subread, version 1.6.4. The differential gene expression analysis (DEG) was assessed with voom+limma in the limma package version 3.40.2 and using R version 3.6.0. Raw library size differences between samples were treated with the weighted trimmed mean method implemented in the edgeR package. Clustering method used is Ward.D2 with correlation distances and principal components analysis. For the differential expression analysis, read counts were converted to log2 counts per million, and the mean-variance relationship was modeled with precision weights using voom approach in limma package. Raw data are accessible at the NCBI Gene Expression Omnibus (GEO) accession code GSE131286.

Intersection of DEG for A673 shRING1B knockdown with those for A673 shEWSR1-FLI1 with accession number GSE61953 (10) was obtained by calculating a delta-score as described by the authors. Absolute FC > 1.25 and 1.5 for RING1B and EWSR1-FLI1 datasets were selected, respectively. Overlaps for positive and negative gene sets were obtained using Vennerable R package and BioVenn. Functional analysis of the intersection between RING1B and EWSR1-FLI1 gene lists was performed in Enrichr. Normalized enrichment scores on A673 and SK-ES1 shRING1B versus shCTRL were obtained with GSEA using the Hallmark gene set collection. GSEA was used to analyze enrichment on the list of 983 down-regulated TSG in tumor samples versus normal tissue from TSGene database (50) (https://bioinfo.uth.edu/TSGene/). Analysis of expression levels for AURKB, CSNK2A1, and MAP2K1 were performed using information from GEO2R GSE7007 for the probes 209464_at, 212075_s_at, and 202670_at, respectively.

Immunohistochemical analyses were performed following standard techniques. The antibodies used are given in table S6. Tumors were fixed in formalin and embedded in paraffin for subsequent processing. Consecutive, sections were deparaffinized, rehydrated, and heated with Epitope Retrieval Solution (pH 6.0) (Novocastra Laboratories). Reactions were developed with Novolink Polymer Detection System (Novocastra Laboratories). Immunoreactivity was visualized by diaminobenzidine, and nuclei were counterstained with hematoxylin. Tissue was then dehydrated with alcohol, permeated with xylene, and mounted with Permount organic mounting solution (Thermo Fisher Scientific). Images were evaluated by a pathologist to select regions of interest and analyzed with the Dotslide Microscope and Olympia Software (Olympus). Similar regions of every sample were selected from every section.

Cells were treated with 1% formaldehyde at room temperature for 10 min, and the cross-linking reaction was stop by adding 500 l glycine (1.25 M). Cells were resuspended in lysis buffer [0.1% SDS, 0.15 M NaCl, 1% Triton X-100, 1 mM EDTA, 20 mM tris (pH 8), and protease inhibitors (1 mg/ml)] and sonicated with Bioruptor Pico (Diagenode) for 10 cycles until chromatin was sheared to an average fragment length of 200 bp. After centrifugation, a small fraction of eluted chromatin was measured with Qubit. Starting with 30 g of sample, immunoprecipitation for each antibody was performed overnight (table S6); 50 l of Dynabeads Protein A (Invitrogen) was then added and incubated for 2 hours at 4C under rotation. Immunoprecipitates were washed once with TSE I [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM tris-HCl (pH 8), and 150 mM NaCl], TSE II [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM tris-HCl (pH 8), and 500 mM NaCl], and TSE III [0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, and 10 mM tris-HCl (pH 8)] and then twice with tris-EDTA buffer. Washed pellets were eluted with 120 l of a solution of 1% SDS and 0.1 M NaHCO3. Eluted pellets were decross-linked for 5 hours at 65C and purified on 50 l of tris-EDTA buffer with the QIAquick PCR Purification Kit (Qiagen). Differences in the DNA content at each binding region (sequences in table S6) from every immunoprecipitation assay were determined by real-time PCR using the ABI 7700 sequence detection system and SYBR Green master mix protocol (Applied Biosystems). Each immunoprecipitation was done in triplicate, and PCR assays were performed using fixed amounts of input and immunoprecipitated DNA. For every amplicon, standard curves to calculate efficiency and melting curves to confirm single amplicons were obtained. The reported data represent real-time PCR values normalized to input DNA and are expressed as percentage (%) of bound/input signal.

Libraries were prepared using the NEBNext Ultra DNA Library Prep from Illumina according to the manufacturers protocol. Briefly, 5 ng of input and ChIP-enriched DNA were subjected to end repair and addition of A bases to 3 ends, ligation of adapters, and USER excision. All purification steps were performed using AgenCourt AMPure XP beads (Qiagen). Library amplification was performed by PCR using NEBNext Multiplex Oligos from Illumina. Final libraries were analyzed using Agilent high sensitivity chip to estimate the quantity and to check size distribution and then were quantified by qPCR using the KAPA Library Quantification Kit (KapaBiosystems) before amplification with Illuminas cBot. Libraries were loaded onto the flow cell sequencer 1 50 on Illuminas HiSeq 2500.

ChIP-seq samples were mapped against the hg19 human genome assembly using BowTie with the option m 1 to discard those reads that could not be uniquely mapped to just one region. A second replicate of RING1B and H2Aub was sequenced to evaluate the statistical significance of the results. Model-based analysis of ChIP-seq (MACS) was run individually on each replicate with the default parameters but with the shift size adjusted to 100 bp to perform the peak calling against the corresponding control sample (51). DiffBind was initially run over the peaks reported by MACS for each pair of replicates of the same experiment to generate a consensus set of peaks (28). Next, DiffBind was run again over each pair of replicates of the same experiment, samples and inputs, to find the peaks from the consensus set that were significantly enriched in both replicates in comparison to the corresponding controls (categories, DBA_CONDITION; block, DBA_REPLICATE; and method, DBA_DESEQ2_BLOCK). DiffBind RING1B peaks with P < 0.05 and H2Aub peaks with P < 0.05 and false discovery rate < 0.00001 were selected for further analysis. The genome distribution of each set of peaks was calculated by counting the number of peaks fitted on each class of region according to RefSeq annotations. Promoter is the region between 2.5 kb upstream and 2.5 kb downstream of the TSS. Genic regions correspond to the rest of the gene (the part that is not classified as promoter), and the rest of the genome is considered to be intergenic. Peaks that overlapped with more than one genomic feature were proportionally counted the same number of times. Each set of target genes was retrieved by matching the ChIP-seq peaks in the region 2.5 kb upstream of the TSS until the end of the transcripts as annotated in RefSeq. Reports of functional enrichments of GO categories were generated using the EnrichR tool. Aggregated plots showing the average distribution of ChIP-seq reads around the summit of each peak were generated by counting the number of reads for each region and then averaging the values for the total number of mapped reads of each sample and the total number of peaks in the particular gene set. To perform the comparison between two sets of peaks, a minimum overlap of one nucleotide was necessary to consider one match. The heatmap displaying the density of ChIP-seq reads 5 kb around the summit of each peak set were generated by counting the number of reads in this region for each individual peak and normalizing this value with the total number of mapped reads of the sample. Peaks on each ChIP heatmap were ranked by the logarithm of the average number of reads in the same genomic region. On the other hand, we separated the single peaks of RING1B into distal and TSS (5 kb around one RefSeq gene) to generate the heatmap of ChIP-seq signal strength of RING1B, EWSR1-FLI1, H3K27me3, H2Aub, and H3K27ac over the two classes of RING1B peaks detected above (distal and TSS). To build our collection of enhancers and promoters, we reanalyzed published ChIP-seq samples of H3K4me1, H3K27ac, H3K27me3, and H3K4me3 in A673 cells (10). H3K27ac and H3K27me3 peaks were used to discriminate between active or repressed regulatory regions. Promoters were defined as ChIP peaks of H3K27 found up to 2.5 kb from the TSS of one gene and enhancers on intergenic areas outside promoters or within gene introns. H3K4me3 was required to be present in promoters but absent in enhancers. We defined four classes of regulatory elements: active enhancers (H3K27ac), active promoters (H3K27ac + H3K4me3), poised enhancers (H3K27me3), and bivalent promoters (H3K27me3 + H3K4me3). The MEME-ChIP tool was used to perform motif-finding analysis of the sequences bound by each factor. The UCSC genome browser was used to generate the screenshots of each group of experiments along the manuscript (52). Raw data, genome-wide profiles, and peaks of each ChIP-seq experiment are accessible at the NCBI GEO accession code GSE131286.

We have determined the composition of 3945 EWSR1-FLI1 biding sites in terms of 15 chromatin states from the segmentations generated by Epigenome Roadmap Consortium (GEO code: GSE61953) for six different cell types: HUVECs (E122), H1 (E003) and H9 ES cells (E008), H1-derived mesenchymal stem cells (E006), BM-derived MSCs (E026), and adipose-derived MSC (E025) (26). The statistical significance of the relative frequency of each stage at every cell type was assessed in comparison to the same value measured along the whole genome, using the Fishers exact test. The R package GenomicRanges from Bioconductor was used for calculations of compositions. Next, to generate the final heatmap, we have grouped certain states for semantic similarity (active TSS category includes active and flanking active TSS states; transcription includes flanking, strong, and weak states; enhancers account for both genic and intergenic; bivalent TSS include also flanking bivalent promoters and PcG repressed include both repressed and weak repressed). Thus, the relative frequencies of the new eight states were recalculated, while quiescent state was discarded from the analysis. Last, the enrichment percentage at a particular stage was calculated as the difference between the relative frequency at the EWSR1-FLI1 ChIP-seq sites minus the relative frequency at the whole genome normalized by the relative frequency at the whole genome again.

In vivo studies were performed after the approval of the Institutional Animal Research Ethics Committee. Athymic nude mice (Envigo) were injected subcutaneously with 4 106 cells for shCTRL#seq1 and shRING1B#seq1 and 2 106 for seq#2. shCTRL cells were resuspended in 200 l of Matrigel (Becton Dickinson) with phosphate-buffered saline and injected into both flanks (5 mice n = 10 for seq#1 and 6 mice, n = 12 for seq#2). The same procedure was performed for the SK-ES1 shRING1B cell line. Tumor growth was monitored three times a week by measuring tumor volume with a digital caliper. Mice were euthanized when tumors reached a size of 2.5 cm in any dimension. Survival curves were calculated using the Kaplan-Meier method and were compared with a log-rank test. At the end of the experiment, tumors were excised; half of each specimen was frozen in liquid nitrogen for RNA extraction, and the other was fixed in 10% formalin for immunohistochemistry experiments.

Acknowledgments: We thank N. Riggi for reagents and technical advice and M. Martnez-Balbs for technical advice and critical reading of the manuscript. We also thank G. Pascual-Pasto, S. Mateo, and M. Suol for technical advice, S. Perez-Jaume for statistical advice, and L. Nonell from the Microarray Analysis Service, Hospital del Mar Medical Research Institute (IMIM, Barcelona) for technical advice. Last, we are grateful to the Band of Parents at Hospital Sant Joan de Du for supporting the overall research activities of the developmental tumor laboratory, PCCB. Funding: S.S.-M. and the project were supported by the Spanish Association Against Cancer (AECC) consolidated groups grant (GCB13131578) consortium. The project also had the support from the Asociacion Pablo Ugarte (APU). E.F.-B. was supported by the Spanish government grant, Instituto de Salud Carlos III (PI16/00245) to J.M. The work in the Di Croce laboratory was supported by grants from the Spanish of Economy, Industry and Competitiveness (MEIC) (BFU2016-75008-P), and Fundacion Vencer El Cancer (VEC). Author contributions: S.S.-M., L.D.C., and J.M. designed the study, conducted experiments, and wrote the manuscript. J.M. supervised all the work. S.S.-M., E.F.-B., M.S.-J., P.T., C.B., E.P., L.H.-P., and D.J.G.-D. performed the experiments. E.B. and S.G. performed all the bioinformatic analysis. I.H.-M., O.M.T., A.M.C., C.L., and E.. provided expertise and feedback. All authors reviewed the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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RING1B recruits EWSR1-FLI1 and cooperates in the remodeling of chromatin necessary for Ewing sarcoma tumorigenesis - Science Advances

Bone Marrow Stem Cells Comments Off on RING1B recruits EWSR1-FLI1 and cooperates in the remodeling of chromatin necessary for Ewing sarcoma tumorigenesis – Science Advances | October 24th, 2020

Global cosmetic skin care market is set to witness a substantial CAGR of 5.5% in the forecast period of 2019- 2026. The report contains data of the base year 2018 and historic year 2017. Increasing self-consciousness among population and rising demand for anti- aging skin care products are the factor for the market growth.

Global Cosmetic Skin Care Market By Product (Anti-Aging Cosmetic Products, Skin Whitening Cosmetic Products, Sensitive Skin Care Products, Anti-Acne Products, Dry Skin Care Products, Warts Removal Products, Infant Skin Care Products, Anti-Scars Solution Products, Mole Removal Products, Multi Utility Products), Application (Flakiness Reduction, Stem Cells Protection against UV, Rehydrate the skins surface, Minimize wrinkles, Increase the viscosity of Aqueous, Others), Gender (Men, Women), Distribution Channel (Online, Departmental Stores and Convenience Stores, Pharmacies, Supermarket, Others), Geography (North America, Europe, Asia-Pacific, South America, Middle East and Africa) Industry Trends and Forecast to 2026

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Market Definition: Global Cosmetic Skin Care Market

Cosmetic skin care is a variety of products which are used to improve the skins appearance and alleviate skin conditions. It consists different products such as anti- aging cosmetic products, sensitive skin care products, anti- scar solution products, warts removal products, infant skin care products and other. They contain various ingredients which are beneficial for the skin such as phytochemicals, vitamins, essential oils, and other. Their main function is to make the skin healthy and repair the skin damages.

Market Drivers:

Market Restraints:

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Geographically, this report is segmented into several key regions, with sales, revenue, market share and growth Rate of industry in these regions, from 2020 to 2027, covering

Global cosmetic skin care market is highly fragmented and the major players have used various strategies such as new product launches, expansions, agreements, joint ventures, partnerships, acquisitions, and others to increase their footprints in this market. The report includes market shares of cosmetic skin care market for Global, Europe, North America, Asia-Pacific, South America and Middle East & Africa.

Few of the major competitors currently working in the global cosmetic skin care market are LOral, Unilever, New Avon Company, Este Lauder Companies, Espa, Kao Corporation, Johnson & Johnson Services, Inc., Procter & Gamble, Beiersdorf, THE BODY SHOP INTERNATIONAL LIMITED, Shiseido Co.,Ltd., Coty Inc., Bo International, A One Cosmetics Products, Lancme, Clinique Laboratories, llc., Galderma Laboratories, L.P., AVON Beauty Products India Pvt Ltd, Nutriglow Cosmetics Pvt. Ltd, Shree Cosmetics Ltd among others.

Cosmetic Skin Care Market: Key Questions Answered in Report

The research study on the Cosmetic Skin Care market offers inclusive insights about the growth of the market in the most comprehensible manner for a better understanding of users. Insights offered in the Cosmetic Skin Care market report answer some of the most prominent questions that assist the stakeholders in measuring all the emerging possibilities.

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Cosmetic Skin Care Market (Covid 19 Impact Analysis) Data Highlighting Major Vendors, Promising Regions, Anticipated Growth Forecast To 2027 - PRnews...

Skin Stem Cells Comments Off on Cosmetic Skin Care Market (Covid 19 Impact Analysis) Data Highlighting Major Vendors, Promising Regions, Anticipated Growth Forecast To 2027 – PRnews… | October 24th, 2020

Real beauty is reflected in your skin. If you want a clearer, younger looking skin, non-surgical cosmetic treatments and some maintenance after that can let you achieve immediately visible and long-lasting results. You dont have to always undergo cosmetic surgery to improve appearance and reduce the signs of ageing. In fact, you can choose non-surgical cosmetic treatments to reduce wrinkles, plump up your lips, smoothen the surface of your skin. We spoke with Dr. Anup Dhir Senior Consultant, Cosmetic Surgeon, Apollo Hospital, Delhi to know more about non-cosmetic procedures that can bring a drastic change in your appearance and here's what the doctor had to say.Non-surgical cosmetic treatments: Broadly, these treatments are of three types. We can use laser and other energy based devices like radiofrequency and ultrasound, we can do injectables like botox, fillers microfat and PRP, or we can do time tested older procedures like chemical peels, dermabrasion and microneedling to rejuvenate the skin.Laser skin resurfacing for wrinkles is very commonly done with carbon dioxide or erbium fractional laser and helps by removing the top layer of the skin and making it look younger. Laser hair reduction is done with lasers for facial and body hair and normally six sittings are needed at monthly intervals.Radiofrequency energy devices like thermage, exilis, e matrix etc. and ultrasound devices like HIFU help in formation of new collagen under the skin by directing energy at a particular level under the skin.

Botox injection for wrinkles -This injection is very commonly used for treatment of facial wrinkles and weakens the muscles which cause wrinkles. It has to be repeated after 4-6 months.

Anti-wrinkle treatment by fillers is again a very popular procedure in which hyaluronic acid fillers are injected into scars and wrinkles. The resulting improvement lasts for 9-15 months.

Fat injection for scars and wrinkles are the gold standard for rejuvenation and in this, your own body fat is sucked, processed and injected in the facial wrinkles. Microfat and nanofat are the types of fat which can be used. The fat has its own stem cells and they help to rejuvenate the skin and improve texture and; help in face lift and rejuvenation of hands also.

PRP skin rejuvenation is done by taking your blood and making platelet rich plasma from it and this is injected in the facial skin and it can also be used in the scalp to reduce the hair fall and help in regrowth.

Dermabrasion involves taking the outer layer of skin with a diamond roller under a local anesthetic and the new skin on healing has less scars. The purpose of surgical dermabrasion is to help diminish the appearance of deeper scars and skin imperfections and smoothen the skin.

Chemical peels use a chemical solution to smoothen the texture of your skin by removing the damaged outer layers. It is one of the least invasive ways to improve the appearance of your skin. Superficial peels with fruit acids like glycolic acid etc. are also called lunchtime peels as they have hardly any down time and can give quick results. As sun exposure, acne, or just getting older can leave your skin tone uneven, wrinkled, spotted or scarred, these peels can help these conditions. They also helps to whiten the skin.

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How to get a beautiful skin without surgery - Times of India

Skin Stem Cells Comments Off on How to get a beautiful skin without surgery – Times of India | October 24th, 2020

Stem cells are biological cells which have the ability to distinguish into specialized cells, which are capable of cell division through mitosis. Amniotic fluid stem cells are a collective mixture of stem cells obtained from amniotic tissues and fluid. Amniotic fluid is clear, slightly yellowish liquid which surrounds the fetus during pregnancy and is discarded as medical waste during caesarean section deliveries. Amniotic fluid is a source of valuable biological material which includes stem cells which can be potentially used in cell therapy and regenerative therapies. Amniotic fluid stem cells can be developed into a different type of tissues such as cartilage, skin, cardiac nerves, bone, and muscles. Amniotic fluid stem cells are able to find the damaged joint caused by rheumatoid arthritis and differentiate tissues which are damaged.

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Medical conditions where no drug is able to lessen the symptoms and begin the healing process are the major target for amniotic fluid stem cell therapy. Amniotic fluid stem cells therapy is a solution to those patients who do not want to undergo surgery. Amniotic fluid has a high concentration of stem cells, cytokines, proteins and other important components. Amniotic fluid stem cell therapy is safe and effective treatment which contain growth factor helps to stimulate tissue growth, naturally reduce inflammation. Amniotic fluid also contains hyaluronic acid which acts as a lubricant and promotes cartilage growth.

With increasing technological advancement in the healthcare, amniotic fluid stem cell therapy has more advantage over the other therapy. Amniotic fluid stem cell therapy eliminates the chances of surgery and organs are regenerated, without causing any damage. These are some of the factors driving the growth of amniotic fluid stem cell therapy market over the forecast period. Increasing prevalence of chronic diseases which can be treated with the amniotic fluid stem cell therapy propel the market growth for amniotic fluid stem cell therapy, globally. Increasing funding by the government in research and development of stem cell therapy may drive the amniotic fluid stem cell therapy market growth. But, high procedure cost, difficulties in collecting the amniotic fluid and lack of reimbursement policies hinder the growth of amniotic fluid stem cell therapy market.

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The global amniotic fluid stem cell therapy market is segmented on basis of treatment, application, end user and geography:

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Rapid technological advancement in healthcare, and favorable results of the amniotic fluid stem cells therapy will increase the market for amniotic fluid stem cell therapy over the forecast period. Increasing public-private investment for stem cells in managing disease and improving healthcare infrastructure are expected to propel the growth of the amniotic fluid stem cell therapy market.

However, on the basis of geography, global Amniotic Fluid Stem Cell Therapy Market is segmented into six key regionsviz. North America, Latin America, Europe, Asia Pacific Excluding China, China and Middle East & Africa. North America captured the largest shares in global Amniotic Fluid Stem Cell Therapy Market and is projected to continue over the forecast period owing to technological advancement in the healthcare and growing awareness among the population towards the new research and development in the stem cell therapy. Europe is expected to account for the second largest revenue share in the amniotic fluid stem cell therapy market. The Asia Pacific is anticipated to have rapid growth in near future owing to increasing healthcare set up and improving healthcare expenditure. Latin America and the Middle East and Africa account for slow growth in the market of amniotic fluid stem cell therapy due to lack of medical facilities and technical knowledge.

Some of the key players operating in global amniotic fluid stem cell therapy market are Stem Shot, Provia Laboratories LLC, Thermo Fisher Scientific Inc. Mesoblast Ltd., Roslin Cells, Regeneus Ltd. etc. among others.

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The Amniotic Fluid Stem Cell Therapy market to invigorate from 2018 to 2026 - TechnoWeekly

Cardiac Stem Cells Comments Off on The Amniotic Fluid Stem Cell Therapy market to invigorate from 2018 to 2026 – TechnoWeekly | October 24th, 2020

This article was reviewed by Russell N. Van Gelder, MD, PhD

Neuronal cell replacement therapies remain a challenge in retinal diseases. Some fish and salamanders have the innate ability to regenerate retinal tissue after injuries and, as Russell N. Van Gelder, MD, PhD, pointed out, if researchers could harness this ability in humans, the possibilities would be great for repairing or replacing damaged tissue in a wide variety of retinal diseases. Stem cells are the key to cell replacement therapies.

Stem cells are cells that have not terminally differentiated and still have the potential to become many types of terminal cells, said Van Gelder, from the Department of Ophthalmology at the University of Washington in Seattle. We all started as embryonic stem cells in the earliest phases of development.

Related: Retinal pathologies challenging to image with current technologies

Van Gelder went on to explain that there are now methods to create equivalently totipotent stem cells from individual induced progenitor stem cells derived from an individuals blood or epithelial cells.

The overarching goal is to create a cell type that needs replacement from a stem cell precursor, he said.

A major achievement in this quest for regenerative ability occurred in 2014 when an entire eye cup was grown from progenitor stem cells.

Van Gelder also described a study1 in which green fluorescent proteinlabeled retinal precursors derived from embryonic stem cells were transplanted into the subretinal space of macaques. Three months after the procedure, the researchers demonstrated that the bolus of cells persisted and had outgrowth of axons that were seen going to the optic nerve and on to the brain.

This result establishes the validity of a stem cell-based approach for doing regenerative medicine in primates, he said.

Related: Persistent retinal detachment associated with retinoblastoma

Replacement therapy hurdlesAs of now, however, no stem cell-based replacement treatment has received FDA approval. The problems preventing establishment of a treatment have been technical in nature and include correct cellular differentiation as well as generating adequate numbers of cells for large transplantation experiments, establishing correct cell polarity and connectivity, and ensuring the safety of these approaches regarding tumor or hamartoma formation, Van Gelder explained.

Managing inflammatory responses is a problem after cell transplantation. He cited a Japanese study2 of individual progenitor cell-derived retinal progenitor cells transplanted subretinally in monkey models.

Even with an immune HLA-matched donor, there was still a marked inflammatory response at the site of the transplantation, Van Gelder said. This and other inflammatory responses will have to be managed for cell transplantation to be successful.Related: Intravitreally injected hRPCs improve vision in retinitis pigmentosa cases

There are regulatory hurdles to clear. The FDA Center for Biologics Evaluation and Research regulates cellular therapy products, human gene therapy products, and certain devices related to cell and gene therapy.

Van Gelder recalled the well-publicized case of transplantation of fat-derived mesenchymal cells into patients eyes, resulting in loss of vision bilaterally. He pointed out that it is important to temper patient expectations regarding these therapies and to ensure that the work is being done with the highest degree of ethical integrity.

While great progress has been made in this field, significant barriers remain to the successful adoption in the clinical setting in the coming years, Van Gelder concluded. The barriers to cell replacement should be overcome.

Read more by Lynda Charters

--

Russell N. Van Gelder, MD, PhDe: russvg@uw.edu Van Gelder has no financial interests in this subject matter. He serves on the advisory committee for the National Eye Institute Audacious Goals Initiative.

--

References

1. Chao JR, Lamba DA, Kiesert TR, et al. Transl Vis Sci Technol. 2017;6:4; doi:10.1167/tvst/6/3/4

2. Fujii S, Sugita S, Futatsugi Y, et al. A strategy for personalized treatment of iPS-retinal immune rejections assessed in cynomolgus monkey models. Int J Mol Sci. 2020;21(9):3077. doi:10.3390/ijms21093077

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Harnessing regeneration of retinal tissues: An option almost within reach - Ophthalmology Times

IPS Cell Therapy Comments Off on Harnessing regeneration of retinal tissues: An option almost within reach – Ophthalmology Times | October 23rd, 2020

Getting up there in years comes with its drawbacks and benefits, and the onset of facial lines and volume loss that comes tends to be at the top of the list as one of its main disadvantages. You can count facial aging right up there with the onslaught of back pain and the occasional grey hair turning into a full head of silver. To soften those where did they come from facial lines and give skin a more youthful glow, these anti-agers target the main offenders: wrinkles and uneven skin tone and texture.

2/8

Glycolipids in Dr. Loretta Intense Replenishing Serum ($70) trash moisture on the skin surface to help hydrate skin while the antioxidant lipochroman combats free radicals and protects from harmful UV light, leaving skin looking plump, smooth and rejuvenated.

3/8

Apply a layer of Augustinus Bader The Face Oil ($230) morning and night. Utilizing Professor Baders TFC8 technology, the oil promotes cellular renewal, which helps smooth skin texture and reduce the look of fine lines and wrinkles.

4/8

The name says it all with Zo Skin Healths Firming Serum ($235). Lightweight and tolerable for even sensitive skin types, this anti-ager includes the brands ZCORE complex which consists of a synthetic tetrapeptideand sweet yellow clover to help strengthen skin laxity. Plant stem cells provide plant stem cell complex provides powerful antioxidant protection while sodium DNA helps stimulate cell repair and reduce inflammation.

5/8

Harnessing the brands signature ingredient, La Prairies Skin Caviar Liquid Lift ($690) blends two types of caviar, Premiere and Absolute, into a milky emulsion to deliver the perfect dose of serum that promises firmer skin and enhanced elasticity.

6/8

Bioeffect Limited Edition EGF Serum ($495) is said to have twice the original EGF formulas anti-aging benefits due to its inclusion of a rare black barley that is grown at the brands state-of-the-art greenhouse in Iceland. The EGF stands for Epidermal Growth Factor, which in this serum is totally plant derived and signals skin cells to prompt collagen and elastin production. The unique bottle was designed by Icelandic artist Shoplifter and is made from black obsidian.

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8 Skin-Boosting, Anti-Aging Treatments for Generation Xers and Beyond - NewBeauty Magazine

Skin Stem Cells Comments Off on 8 Skin-Boosting, Anti-Aging Treatments for Generation Xers and Beyond – NewBeauty Magazine | October 23rd, 2020

Augustinus Bader on his revolutionary approach to skincare

The mind behind the most coveted products in beauty discusses thescience behind the brand

When Augustinus Bader first launched The Cream in 2018, it was hailed as a miracle. In a matter of weeks, it could transform any skin type within any age range, dispelling wrinkles, redness, dryness, scaring, visible pores, sagginess, and practically every other skin concern it would usually take a shelf load of serums to combat. While miraculous, magical, and other mystical attributions caneasily, and quite fairly, be applied to Augustinus Bader products, the real genius of the brand comes down to pure science.

The Augustinus Bader skincarebrand was the by-product of its namesakes development of medical-grade cream, which could heal severe burn injuries to an extent that was previously only possible through skin grafts. Professor Bader, a stem cell and biomedical scientist at Leipzig University, was hoping to get the cream backed by a pharmaceutical company but, in the words of his business partner Charles Rosier, clinical trials cost tens of tens of millions of dollarsand the majority of accidents around burns happen to children, often in third-world countries. For a pharmaceutical company, when the outcome is not necessarily the most profitable outcome, theres less interest.

Inspired to make Baders cream widely available, Rosier encouraged the Professor to translate the principles of his burn cream into skincare. In my mind, I thought, if we create a cream thats superior to whats on the market and its a big success, then he can focus on is research and we can finance the clinical trial.

Baders cream centred around one, revolutionary hypothesisthat the body already possesses all of the stem cells it needs to regenerate itself. The problem, when it comes to the skins inability to heal from severe injuries or just the everyday effects of ageing, is that the bodys ability to trigger those regenerative cells has been impeded.

Bader developed this hypothesis based on two observations. First, that the size of the wound affects the bodys ability to heal. Asmall paper cut heals quickly, while a large scale burn takes time to heal and often leaves scar tissue. Secondly, the body automatically knows where the site of an injury is. When you cut your left hand, your body immediately starts sending cells to the area of the cut so that the skin can rebuild. Yet,the same tissue would never rebuild on your right hand because it rebuilt on your left. Only where there is a wound is the body rebuilding.

In Baders words, If the cut is super small, you would have a small distance between the edges of the cut skin and the cells can still communicate over this small distances through the hand, and would close the wound. But if you burn your hand, the cells would be dying and the signal response cannot arrive at this injury. The response is totally different, the small cut heals perfectly, while on the other side the big injury kills this confirmation.

So the basis of [my] hypothesis is that this is probably just the absence of specific molecules that cannot arrive to the site of the injury because cells are dying or are blocked. So many, many years ago I started trying to find solutions to this problem because genetically speaking were the same human being, why would we have these limitations, why would we have these problems? It doesnt make any sense.

I thought, why not try to replace what the cells would be doing if they were present? That triggering complex, which singles the cells to respond to the injury, or, when it comes to skincarewrinkles, is the secret, miracle likeelement of Baders cream.

Unlike most skincare, which just changes the outside surface of the skin, Baders skincare works from the inside out, transforming the bodys internal, cellular communication for exterior changes. I think ageing is just a lack of repair, a lack of regeneration. Skin is a living organism, which has to be remodelled, meaning repaired a little bit everyday. But you can accelerate this repair lead.

Theres something super, super sensitive inside of you, which are these cells that sense the microenvironment and respond to the need. So the cream, in a way, is only a toolbox, which helps your stem cells when they sense this need to interact more appropriately.

This new approach to the effects of ageing is a revolution in skincare that, no doubt, heralds the beginning of a new science-driven, cellular-focused trend in the industry.

This year, Bader has launched a number of additional products to his line beyond The Cream and its companion The Rich Cream. The new additions include a Cleansing Gel, Face Oil, Body Cream and, as of today, Cleansing Balm with more releases set for the next year.

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Augustinus Bader on his revolutionary approach to skincare - Wallpaper*

Skin Stem Cells Comments Off on Augustinus Bader on his revolutionary approach to skincare – Wallpaper* | October 23rd, 2020

Top notch ingredients are vital when it comes to creams. We chatted with skin care pros to see which ingredients are the best . This helped us pick out the products with the most oomph.

We also factored in:

Pricing guide

Night creams def have a rep for being expensive and some totally are. You should expect to pay more for extra bells and whistles (e.g. designer brands, fancy packaging, etc.). But thats definitely not the case percent of the time. You can find awesome, dermatologist-recommended products for around $10.

This guide will help you pick the best cream for your skin and budget:

$ = $10$20$$ = $25$50$$$ = $51$75$$$$ = over $76

Whether youre looking for a simple cream that gives your skin a glowy boost or a powerful cream for more mature skin to help reduce fine lines, theres a cream for you. Here are the top 22 night creams for every need.

Price: $$$$

Designed for all skin types, this lightweight cream uses retinol to reduce the appearance of lines. Hyaluronic acid delivers hydration and improves skins tone and texture. It also has niacinamides and picolinamides that support your skins natural barrier and lock in moisture.

Cons: Some peeps with sensitive skin said it caused irritation.

Buy Murad Retinol Youth Renewal Night Cream online.

Price: $

Unlike some heavy duty hydrators, this cream is oil-free and wont clog pores. You can use it day and night without worrying about pesky pimples.

It has the benefits of anti-aging while being lightweight enough to not trigger acne, says dermatologist Erum Ilyas, MD, MBE, FAAD. If youre looking for a cream but dont want to risk breakouts, this is a nice one to try.

Cons: It might not be hydrating enough for dry skin.

Buy OLAY Total Effects 7-in-1 Anti-Aging Moisturizer online.

Price: $$

Found: An overnighter that fights the signs of aging and keeps breakouts at bay. Retinol helps plump skin to reduce the appearance of lines and wrinkles. Salicylic and lactic acids keep bacteria from clogging pores and causing breakouts.

Cons: Salicylic acid can be drying.

Buy Arcona PM Blemish Lotion online.

Price: $$

This concentrated balm harnesses the power of colloidal oatmeal and sweet almond oil to soothe itchy, inflamed skin. It promotes a smoother and more even skin texture and can help reduce redness. Its even safe to use around your eyes and on your lids.

Cons: Some users found the rich texture to be a bit greasy.

Buy Skinfix Eczema Dermatitis Face Balm online.

Price: $$

This cream delivers heavy duty hydration to fight ashiness (thanks, avocado and shea butter). The vitamin C can help combat hyperpigmentation from exposure to UV rays (which is more likely in darker skin).

Cons: It might trigger breakouts in oily or acne-prone skin.

Buy Eve Hansen Vitamin C Night Cream online.

Price: $$

This super hydrating treatment straddles the line between cream and mask. Ingredients like squalene, glycerin, and fountain plant quench parched skin. It also helps protect the skins natural barrier to keep moisture in.

Cons: Some users complain that the texture is too thick to the point of being straight up sticky.

Buy Kiehls Ultra Facial Overnight Hydrating Masque online.

Price: $$$$

Dermatologist Deborah Longwill, DO, FAOCD, counts this potent potion as a current fave.

It combines the anti-aging ingredient resveratrol with antioxidant-rich ingredients like glycoin and ectoin, she explains.

These ingredients help shield your skin from environmental stresses. They also work to enhance elasticity, improve texture, and hydrate cells.

Cons: Its on the spendy side.

Buy Doctors Daughter Extremolyte Stem Cell Serum online.

Price: $$$

This hydrating-but-not-overly-heavy cream nourishes and plumps skin with ingredients like ceramides and hyaluronic acid. Oh, and its been clinically tested to reduce fine lines, dryness, and loss of firmness in just 7 nights.

Cons: Steer clear if youre not a lavender fan.

Buy IT Cosmetics Confidence in Your Beauty Sleep Night Cream online.

Price: $$

Does added fragrance irritate your skin? Same. Thankfully, this non-irritating cream that gets the job done. Its also loaded with vitamin E which fights redness and inflammation.

Cons: This cream is definitely on the thick side. It might feel heavy on oily skin.

Buy Olay Regenerist Night Recovery Anti-Aging Face Moisturizer online.

Price: $

Retinols a go-to ingredient for minimizing the appearance of fine lines thanks to its ability to protect the skin-plumping protein collagen.

It also has hyaluronic acid, a moisturizer to help prevent irritation and dryness that may be a better option for those with dry or sensitive skin, says dermatologist Susan A. Bard, MD.

Cons: Some users report experiencing redness or rashes.

Buy Neutrogena Rapid Wrinkle Repair Night Moisturizer online.

Price: $$$

This cream uses bakuchiol, a natural retinol alternative. Thats good news if you have sensitive skin.

Its a functional analog of retinol meaning it has the same effect, with one huge advantage: Its less irritating because its also an anti-inflammatory agent, Ilyas says.

Cons: Its got a strong peachy scent that you might love or hate.

Buy OLEHENRIKSEN Goodnight Glow Retin-Alt Sleeping Crme online.

Price: $

Ahhh. Heres a cooling gel cream made with licochalcone, a licorice-based skin soother. It fights redness and irritation in folks with sensitive, rosacea-prone skin. The creams noncomedogenic so it wont clog your pores either.

Cons: This stuffs very gentle. But it still might be too strong on super sensitive skin. Def do a patch test before slathering it all over your face.

Buy Eucerin Redness Relief Night Cream online.

Price: $

Bard loves recommending this simple, no-frills wrinkle fighter to patients. Its inexpensive, easy to find at most drugstores, and it works.

It contains retinol which helps improve fine lines and wrinkles, stimulate collagen production and decrease pigmented spots, she says.

Cons: The retinol in this formula is designed for daily use. But its still worth starting off gradually and work your way up. This gives your skin time to adjust.

Buy RoC Retinol Correxion Deep Wrinkle Night Cream online.

Price: $$

Lotus and peach extract fight oxidative stress and keep your skin looking glowy. But what really sets this lightweight cream apart is the floral peach aroma that comes wafting out the second you open the jar. Another perk: Its good for all skin types.

Cons: Its not formulated to fight fine lines or wrinkles.

Buy Lotus Youth Preserve Dream Face Cream online.

Price: $$$

Grease is not the word here. The gel formula delivers hydration but its still light and cooling. Its got niacinamide, viniferine, and natural pearlizers to fight the appearance of dark spots even out skin tone.

Cons: Some peeps said it didnt brighten their skin.

Buy Caudalie Vinoperfect Instant Brightening Moisturizer online.

Price: $$$

TBH the whole women vs. men products thing is silly. Right? But this cream feels a bit more manly thanks to the neutral packaging and woodsy scent. It fights fine lines and wrinkles with retinol and uses the antioxidant ferulic acid to combat dark spots and sun damage.

Cons: The heavy-duty retinol can be a little harsh especially if your skins not used to it.

Buy Dr. Dennis Gross Ferulic + Retinol Moisturizer online.

Price: $$

Glycolic acid is great at reducing the appearance of dark spots because it can suppress the production of melanin. The acid improves skins elasticity and boosts firmness too. So its an all-around awesome fountain of youth-kinda option.

Cons: Its a serum. If youre looking for hydration, youll still want to layer a moisturizer over top.

Buy Bolden Nighttime Repair Serum with 10% Glycolic Acid online.

Price: $$

Vitamin C and collagen are your eyes BFF. They brighten and plump the delicate skin around your peepers. This ones got both and a little goes a long way.

Cons: Its thick and rich. So it might clog your pores if it ends up on your T-zone.

Buy OLEHENRIKSEN Banana Bright Eye Cream online.

Price: $$$

This certified-organic cream boasts vitamin C, fruit stem cells, grape-seed oil, and squalene. It will brighten and hydrate without the use of parabens, petroleum, sulfates, pesticides, or phthalates.

Cons: The grape-seed oil might be too much for oily or acne-prone skin.

Buy Juice Beauty Stem Cellular Anti-Wrinkle Overnight Cream online.

Price: $

You can legit get amazing results from a night cream without spending megabucks. This dermatologist-developed moisturizer plumps and renews skis with a peptide complex. It also restores the skins natural barrier with essential ceramides. Plus its not greasy!

Cons: This is definitely a utilitarian option. If you love extras like scents or pretty packaging, skip it.

Buy CeraVe Skin Renewing Night Cream online.

Price: $

The suns UV rays can seriously stress your skin. This can cause dark spots, discoloration, and fine lines. But ingredients like green tea and vitamin C help fight sun-induced stress. This hydrating cream delivers both.

Cons: The packaging looks like it came from 1995, which, depending on what youre going for might ruin your #shelfie. (Or maybe not.)

Buy LILY SADO TEA+C Green Tea + Vitamin C Moisturizer online.

Price: $

Snow mushroom and sodium hyaluronate deliver mega moisture, while soothing lavender oil and chamomile extract help you chill and unwind. After anointing yourself with this vegan lotion, you might just wanna close your eyes and doze off.

Cons: You wont get as much anti-aging action here.

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22 Best Night Creams 2020 for All Skin Types and Concerns - Greatist

Skin Stem Cells Comments Off on 22 Best Night Creams 2020 for All Skin Types and Concerns – Greatist | October 23rd, 2020

TUCSON, Ariz., Oct. 21, 2020 /PRNewswire/ --Registration is now open for the World Cord Blood Day 2020 virtual conference (register free on Eventbrite) featuring world renown cord blood transplant doctors and cellular therapy researchers. To be held on November 17th, the virtual conference will provide an opportunity for healthcare professionals, expectant parents, and students to learn about life-saving cord blood stem cells via a mix of livestream and on-demand sessions. The public is also invited to participate in a wide variety of free educational events being held around the globe by WCBD Official Participants (see listings on http://www.WorldCordBloodDay.org).

Attendees of the virtual conference will learn how cord blood has been used in more than 40,000 stem cell transplants since 1988 to treat over 80 life-threatening diseases including leukemia, sickle cell anemia, thalassemia, and lymphoma. Ground-breaking research will also be presented by scientists who are discovering cord blood's full potential in CAR-NK immunotherapy, the emerging field of regenerative medicine to potentially treat autism, cerebral palsy, Covid-19 related MIS-C and more. Keynote presentations will be made by Dr. Joanne Kurtzberg (Duke Department of Pediatrics, Duke Center for Autism and Brain Development), Dr. Katy Rezvani (MD Anderson Cancer Center), Dr. Jonathan Gutman (University of Colorado), Dr. Leland Metheny (Case Western Reserve University), and Monroe Burgess (Quick Specialized Healthcare Logistics). Dr. Moshe Israeli (Rabin Medical Center) will lead the opening session on HLA matching and cord blood.

In addition, a panel of industry experts will discuss how cord blood has come to the forefront during the Covid-19 pandemic. Increasingly, stem cells transplant doctors are using cord blood units collected well before the pandemic and now available for immediate use. Attendees will also hear from Dr. David Hall and Vanessa Yenson, who both beat cancer thanks to cord blood transplants.

To view the full agenda, please visit: https://www.worldcordbloodday.org/online-medical-conference-agenda-wcbd-2020.html

Organized and hosted by Save the Cord Foundation (501c3 non-profit), this year's event is officially sponsored by Quick Specialized Healthcare Logistics. "We're proud to be a sponsor of World Cord Blood Day for the fourth year in a row. This year is sure to be very informative and exciting, providing the latest information from some of the industry's top doctors and researchers. We're humbled to play a role in the research and development of cord blood derivative therapies by providing logistics supply chain solutions to cord blood, biotech and pharmaceutical companies worldwide," said David Murphy, Executive VP of Quick's Life Science Division.

Inspiring Partners this year include the Cord Blood Association (CBA), Be the Match (NMDP), World Marrow Donor Association (WMDA-Netcord), AABB Center for Cellular Therapy and Foundation for the Accreditation of Cellular Therapy (FACT).

Visit http://www.WorldCordBloodDay.org to learn how you can participate and/or host an event. Join us on social media using the hashtags: #WCBD20 and #WorldCordBloodDay.

About Save the Cord Foundation

Save the Cord Foundation (a 501c3 non-profit) was established to advance cord blood education. The Foundation provides non-commercial information to parents, health professionals and the public regarding methods for saving cord blood, as well as current applications using cord blood and the latest research. Learn more at http://www.SaveTheCordFoundation.org.

About Quick Specialized Healthcare Logistics

Quick is the trusted logistics leader serving the Healthcare and Life Science community for almost 40 years. Quick safely transports human organs and tissue for transplant or research, blood, blood products, cord blood, bone marrow, medical devices, and personalized medicine, 24/7/365. Quick's specially trained experts work with hospitals, laboratories, blood banks and medical processing canters, and utilize the safest routes to ensure integrity, temperature control and chain of custody throughout the transportation process. Learn more at http://www.quickhealthcare.aero.

Media Contact:Charis Ober[emailprotected]520-419-0269

SOURCE Save the Cord Foundation

http://www.SaveTheCordFoundation.org

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World Cord Blood Day 2020 Speakers to Present Revolutionary CAR-NK Cell Therapy, Potential Treatments for Covid-19 Related MIS-C and Advantages of...

Bone Marrow Stem Cells Comments Off on World Cord Blood Day 2020 Speakers to Present Revolutionary CAR-NK Cell Therapy, Potential Treatments for Covid-19 Related MIS-C and Advantages of… | October 23rd, 2020

SOMERSET, N.J. and NEW YORK, Oct. 22, 2020 (GLOBE NEWSWIRE) -- Catalent (NYSE: CTLT), the leading global provider of advanced delivery technologies, development, and manufacturing solutions for drugs, biologics, cell and gene therapies, and consumer health products, and BrainStorm Cell Therapeutics Inc. (NASDAQ: BCLI), a leading developer of cellular therapies for neurodegenerative diseases, today announced an agreement for the manufacture of NurOwn, BrainStorms autologous cellular therapy being investigated for the treatment of amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease or motor neuron disease.

NurOwn induces mesenchymal stem cells (MSCs) to secrete high levels of neurotrophic factors (NTFs) known to promote the survival of neurons and neuroprotection. The therapy has received Fast Track status from the U.S. FDA for ALS and has also been granted Orphan Drug Status for ALS by both the FDA and the European Medicines Agency. BrainStorm is currently completing a 200-patient, double-blind, placebo-controlled, repeat-dosing NurOwn Phase 3 study in the U.S.

As part of its commitment, Catalent will undertake the transfer of the manufacturing process to, and provide future CGMP clinical supply of NurOwn from, its new, 32,000 square-foot cell therapy manufacturing facility in Houston, Texas. On completion of the clinical trials and in anticipation of potential approval of NurOwn, the companies will look to extend the partnership to include commercial supply from the Houston facility.

We are proud to have a partner in Catalent whose excellence in manufacturing quality therapies will support commercial supply of NurOwn, said Chaim Lebovits, Chief Executive Officer of BrainStorm Cell Therapeutics. We know that ALS patients are in urgent need of a new treatment option. If NurOwn is successful in the current clinical trials, this agreement will be integral to ensuring rapid access for patients.

Manja Boerman, Ph.D., President, Catalent Cell & Gene Therapy, said, Our experience in cell therapy development, and the manufacturing capabilities that our newly constructed, state-of-the-art facility in Houston offers, position us to best support BrainStorm, with its leading therapeutic candidate for ALS treatment. We look forward to partnering with BrainStorm and providing our stem cell manufacturing expertise as we work to optimize production and streamline the products path towards commercial launch.

About Catalent Cell & Gene Therapy

With deep experience in viral vector scale-up and production, Catalent Cell & Gene Therapy is a full-service partner for adeno-associated virus (AAV) and lentiviral vectors, and CAR-T immunotherapies. When it acquired MaSTherCell, Catalent added expertise in autologous and allogeneic cell therapy development and manufacturing to position it as a premier technology, development and manufacturing partner for innovators across the entire field of advanced biotherapeutics. Catalent has a global cell and gene therapy network of dedicated, large-scale clinical and commercial manufacturing facilities, and fill-finish and packaging capabilities located in both the U.S. and Europe. An experienced partner, Catalent Cell & Gene Therapy has worked with industry leaders across 70+ clinical and commercial programs.

About Catalent

Catalent is the leading global provider of advanced delivery technologies, development, and manufacturing solutions for drugs, biologics, cell and gene therapies, and consumer health products. With over 85 years serving the industry, Catalent has proven expertise in bringing more customer products to market faster, enhancing product performance and ensuring reliable global clinical and commercial product supply. Catalent employs approximately 14,000 people, including around 2,400 scientists and technicians, at more than 45 facilities, and in fiscal year 2020 generated over $3 billion in annual revenue. Catalent is headquartered in Somerset, New Jersey. For more information, visit http://www.catalent.com

More products. Better treatments. Reliably supplied.

About NurOwn

NurOwn (autologous MSC-NTF) cells represent a promising investigational therapeutic approach to targeting disease pathways important in neurodegenerative disorders. MSC-NTF cells are produced from autologous, bone marrow-derived mesenchymal stem cells (MSCs) that have been expanded and differentiated ex vivo. MSCs are converted into MSC-NTF cells by growing them under patented conditions that induce the cells to secrete high levels of neurotrophic factors. Autologous MSC-NTF cells can effectively deliver multiple NTFs and immunomodulatory cytokines directly to the site of damage to elicit a desired biological effect and ultimately slow or stabilize disease progression. BrainStorm has fully enrolled a Phase 3 pivotal trial of autologous MSC-NTF cells for the treatment of amyotrophic lateral sclerosis (ALS). BrainStorm also received U.S. FDA acceptance to initiate a Phase 2 open-label multicenter trial in progressive MS and enrollment began in March 2019.

About BrainStorm Cell Therapeutics Inc.

BrainStorm Cell Therapeutics Inc. is a leading developer of innovative autologous adult stem cell therapeutics for debilitating neurodegenerative diseases. The Company holds the rights to clinical development and commercialization of the NurOwn technology platform used to produce autologous MSC-NTF cells through an exclusive, worldwide licensing agreement. Autologous MSC-NTF cells have received Orphan Drug status designation from the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the treatment of amyotrophic lateral sclerosis (ALS). BrainStorm has fully enrolled a Phase 3 pivotal trial in ALS (NCT03280056), investigating repeat-administration of autologous MSC-NTF cells at six U.S. sites supported by a grant from the California Institute for Regenerative Medicine (CIRM CLIN2-0989). The pivotal study is intended to support a filing for U.S. FDA approval of autologous MSC-NTF cells in ALS. BrainStorm also recently received U.S. FDA clearance to initiate a Phase 2 open-label multicenter trial in progressive multiple sclerosis (MS). The Phase 2 study of autologous MSC-NTF cells in patients with progressive MS (NCT03799718) completed enrollment inAugust 2020. For more information, visit the company's website at http://www.brainstorm-cell.com.

Safe-Harbor Statement

Statements in this announcement other than historical data and information, including statements regarding future clinical trial enrollment and data, constitute "forward-looking statements" and involve risks and uncertainties that could cause BrainStorm Cell Therapeutics Inc.'s actual results to differ materially from those stated or implied by such forward-looking statements. Terms and phrases such as "may", "should", "would", "could", "will", "expect", "likely", "believe", "plan", "estimate", "predict", "potential", and similar terms and phrases are intended to identify these forward-looking statements. The potential risks and uncertainties include, without limitation, BrainStorm's need to raise additional capital, BrainStorm's ability to continue as a going concern, regulatory approval of BrainStorm's NurOwn treatment candidate, the success of BrainStorm's product development programs and research, regulatory and personnel issues, development of a global market for our services, the ability to secure and maintain research institutions to conduct our clinical trials, the ability to generate significant revenue, the ability of BrainStorm's NurOwn treatment candidate to achieve broad acceptance as a treatment option for ALS or other neurodegenerative diseases, BrainStorm's ability to manufacture and commercialize the NurOwn treatment candidate, obtaining patents that provide meaningful protection, competition and market developments, BrainStorm's ability to protect our intellectual property from infringement by third parties, heath reform legislation, demand for our services, currency exchange rates and product liability claims and litigation,; and other factors detailed in BrainStorm's annual report on Form 10-K and quarterly reports on Form 10-Q available athttp://www.sec.gov. These factors should be considered carefully, and readers should not place undue reliance on BrainStorm's forward-looking statements. The forward-looking statements contained in this press release are based on the beliefs, expectations and opinions of management as of the date of this press release. We do not assume any obligation to update forward-looking statements to reflect actual results or assumptions if circumstances or management's beliefs, expectations or opinions should change, unless otherwise required by law. Although we believe that the expectations reflected in the forward-looking statements are reasonable, we cannot guarantee future results, levels of activity, performance or achievements.

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Catalent and BrainStorm Cell Therapeutics Announce Partnership for the Manufacture of Mesenchymal Stem Cell Platform Therapy NurOwn - GlobeNewswire

Bone Marrow Stem Cells Comments Off on Catalent and BrainStorm Cell Therapeutics Announce Partnership for the Manufacture of Mesenchymal Stem Cell Platform Therapy NurOwn – GlobeNewswire | October 23rd, 2020

NEW YORK, Oct. 22, 2020 (GLOBE NEWSWIRE) -- Mesoblast Limited (Nasdaq:MESO; ASX:MSB), global leader in allogeneic cellular medicines for inflammatory diseases, today announced that a randomized, controlled study of remestemcel-L delivered by an endoscope directly to the areas of inflammation and tissue injury in up to 48 patients with medically refractory Crohns disease and ulcerative colitis has commenced at Cleveland Clinic.

Mesoblast Chief Medical Officer Dr Fred Grossman said: Inflammation of the gut in Crohns disease and ulcerative colitis closely resembles the most severe manifestation of advanced-stage, life-threatening acute graft versus host disease (aGVHD). Mesoblasts objective is to confirm the potential for remestemcel-L to induce luminal healing and early remission in a wider spectrum of diseases with severe inflammation of the gut, in addition to steroid-refractory aGVHD.

Mesenchymal stem cells (MSCs) promote healing of inflamed gut tissue by downregulating gut mucosal effector T-cell activity and promoting regulatory T-cell formation.1 MSCs have been tested in clinical trials of Crohns disease using two different modalities: intravenous infusions of MSCs to treat the primary inflammation of Crohns disease and local injections of MSCs to treat fistulae complicating Crohns disease.

A third modality, endoscopic delivery of MSCs, has been successful in preclinical experimental models of colitis, reducing the excessive cytokine storm in the inflamed gut and resulting in tissue healing.2-3 The study at Cleveland Clinic will be the first in humans using local delivery of MSCs in the gut, and will enable Mesoblast to compare clinical outcomes using this delivery method with results from an ongoing randomized, placebo-controlled trial in patients with biologic-refractory Crohns disease where remestemcel-L was administered intravenously.

The studys lead investigator Dr Amy L. Lightner, Associate Professor of Surgery in the Department of Colon and Rectal Surgery at Cleveland Clinic, stated: We are aiming to establish a new treatment paradigm by administering remestemcel-L at one of two escalating doses, or placebo, directly to inflamed gut tissue in patients with medically refractory Crohns disease and ulcerative colitis, both highly debilitating conditions with significant, unmet medical needs.

According to recent estimates, more than three million people (1.3%) in the US alone have inflammatory bowel disease, with more than 33,000 new cases of Crohns disease and 38,000 new cases of ulcerative colitis diagnosed every year.4-6 Despite recent advances, approximately 30% of patients are primarily unresponsive to anti-TNF agents and even among responders, up to 10% will lose their response to the drug every year. Up to 80% of patients with medically-refractory Crohns disease eventually require surgical treatment of their disease,7 which can have a devastating impact on quality of life.

References1.Mayne C and Williams C. Induced and natural regulatory T cells in the development of inflammatory bowel disease. Inflamm Bowel Dis 2013; 19: 17721788.2.Molendijk I et al. Intraluminal Injection of Mesenchymal Stromal Cells in Spheroids Attenuates Experimental Colitis. Journal of Crohn's and Colitis, 2016, 9539643.Pak S eta al. Endoscopic Transplantation of Mesenchymal Stem Cell Sheets in Experimental Colitis in Rats. Scientific Reports | (2018) 8:11314 | DOI:10.1038/s41598-018-296174.CDC Facts and Figures 20155.Globaldata Pharmapoint 20186.Dahlhamer JM, MMWR Morb Mortal Wkly Rep. 2016;65(42):11661169.7.Crohns and Colitis Foundation

About Remestemcel-LMesoblasts lead product candidate, remestemcel-L, is an investigational therapy comprising culture-expanded mesenchymal stem cells derived from the bone marrow of an unrelated donor. It is administered to patients in a series of intravenous infusions. Remestemcel-L is thought to have immunomodulatory properties to counteract severe inflammatory processes by down-regulating the production of pro-inflammatory cytokines, increasing production of anti-inflammatory cytokines, and enabling recruitment of naturally occurring anti-inflammatory cells to involved tissues.

About MesoblastMesoblast Limited (Nasdaq:MESO; ASX:MSB) is a world leader in developing allogeneic (off-the-shelf) cellular medicines. The Company has leveraged its proprietary mesenchymal lineage cell therapy technology platform to establish a broad portfolio of commercial products and late-stage product candidates. Mesoblast has a strong and extensive global intellectual property (IP) portfolio with protection extending through to at least 2040 in all major markets. The Companys proprietary manufacturing processes yield industrial-scale, cryopreserved, off-the-shelf, cellular medicines. These cell therapies, with defined pharmaceutical release criteria, are planned to be readily available to patients worldwide.

Remestemcel-L is being developed for inflammatory diseases in children and adults including steroid-refractory acute graft versus host disease and moderate to severe acute respiratory distress syndrome. Mesoblast is completing Phase 3 trials for its product candidates for advanced heart failure and chronic low back pain. Two products have been commercialized in Japan and Europe by Mesoblasts licensees, and the Company has established commercial partnerships in Europe and China for certain Phase 3 assets.

Mesoblast has locations in Australia, the United States and Singapore and is listed on the Australian Securities Exchange (MSB) and on the Nasdaq (MESO). For more information, please see http://www.mesoblast.com, LinkedIn: Mesoblast Limited and Twitter: @Mesoblast

Forward-Looking StatementsThis announcement includes forward-looking statements that relate to future events or our future financial performance and involve known and unknown risks, uncertainties and other factors that may cause our actual results, levels of activity, performance or achievements to differ materially from any future results, levels of activity, performance or achievements expressed or implied by these forward-looking statements. All statements other than statements of historical fact are forward-looking statements, which are often indicated by terms such as anticipate, believe, could, estimate, expect, goal, intend, likely, look forward to, may, plan, potential, predict, project, should, will, would and similar expressions and variations thereof. We make such forward-looking statements pursuant to the safe harbor provisions of the Private Securities Litigation Reform Act of 1995 and other federal securities laws. Forward-looking statements should not be read as a guarantee of future performance or results, and actual results may differ from the results anticipated in these forward-looking statements, and the differences may be material and adverse. The risks, uncertainties and other factors that may impact our forward-looking statements include, but are not limited to: statements about the initiation, timing, progress and results of Mesoblast and its collaborators clinical studies; Mesoblast and its collaborators ability to advance product candidates into, enroll and successfully complete, clinical studies; the timing or likelihood of regulatory filings and approvals; and the pricing and reimbursement of Mesoblasts product candidates, if approved; the potential benefits of strategic collaboration agreements and Mesoblasts ability to maintain established strategic collaborations; Mesoblasts ability to establish and maintain intellectual property on its product candidates and Mesoblasts ability to successfully defend these in cases of alleged infringement. You should read this press release together with our risk factors, in our most recently filed reports with the SEC or on our website. Uncertainties and risks that may cause Mesoblasts actual results, performance or achievements to be materially different from those which may be expressed or implied by such statements, and accordingly, you should not place undue reliance on these forward-looking statements. Unless required by law, we do not undertake any obligations to publicly update or revise any forward-looking statements, whether as a result of new information, future developments or otherwise.

Release authorized by the Chief Executive.

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Randomized Controlled Study Using Direct Injection of Remestemcel-L Into Inflamed Gut of Patients With Crohn's Disease and Ulcerative Colitis -...

Bone Marrow Stem Cells Comments Off on Randomized Controlled Study Using Direct Injection of Remestemcel-L Into Inflamed Gut of Patients With Crohn’s Disease and Ulcerative Colitis -… | October 23rd, 2020

The development of tyrosine kinase inhibitors (TKIs) has revolutionized the treatment of chronic myeloid leukemia (CML).1,2 However, despite effectively inducing remission and prolonging survival in patients with CML, TKI therapy does not eradicate leukemia stem cells (LSCs), which are responsible for drug resistance, relapse, and disease progression.2 Given recent changes to the treatment paradigm, updated clinical practice guidelines are essential to ensure optimal clinical care is provided.2

The British Society for Haematology (BSH) published a guideline update for the investigation and management of CML in adults and children in the British Journal of Haematology.1 Lead author of the guidelines, Graeme Smith, MD, of St Jamess University Hospital in the United Kingdom, and coauthors, developed the evidence-based recommendations to provide clinical practitioners with clear guidance on the diagnosis and treatment of adults and children with CML (Tables 1 and 2).

Diagnosis and Key Investigations

The diagnosis of CML is established based on findings from a peripheral blood smear and bone marrow aspirate showing positivity for BCR-ABL1, and the presence of the Philadelphia (Ph) chromosome. The Ph chromosome, or a variant, is present in approximately 95% of CML cases. Other cases include a cryptic BCR-ABL1 fusion, commonly detected by reverse transcriptase polymerase chain reaction (RT-PCR), or fluorescence in situ hybridization (FISH). Additional findings from bone marrow aspirate include the presence of other cytogenetic abnormalities, including isochromosome 17q or trisomy 19, and trisomy 8, suggesting a higher risk of progression to accelerated phase or blast crisis in adults.

Table 1. Selected Recommendations by the BSH Guideline Panel on the Diagnosis of CML1

Table 2. ELTS Score Calculation1

This article originally appeared on Hematology Advisor

Excerpt from:
British Society for Haematology Guideline Update for the Diagnosis and Management of Chronic Myeloid Leukemia - Cancer Therapy Advisor

Bone Marrow Stem Cells Comments Off on British Society for Haematology Guideline Update for the Diagnosis and Management of Chronic Myeloid Leukemia – Cancer Therapy Advisor | October 23rd, 2020

The combination of the BCL2 inhibitor venetoclax with a hypomethylating agent (HMA) may be a treatment option for patients who develop acute myeloid leukemia (AML) secondary to a myeloproliferative neoplasm (MPN), according to results of a small, retrospective cohort study published in Leukemia Research.1

The Philadelphia chromosome-negative MPNs, which include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are clonal disorders resulting in the proliferation of myeloid cells in the bone marrow. Both PV and ET can progress to secondary myelofibrosis which, along with PMF, can progress to secondary AML, also known as MPN-blast phase (MPN-BP).

Furthermore, MPN-BP, defined in this study as being associated with peripheral or bone marrow blasts of at least 20%, is not sensitive to intensive chemotherapy, and clinical outcomes for patients with this disease are very poor. Median overall survival is only approximately 3 to 5 months, and allogeneic hematopoietic stem cell transplantation (HSCT) is considered the only curative option for these patients.

Venetoclax in combination with an HMA, azacitidine, decitabine, or low-dose cytarabine recently received regular US Food and Drug Administration (FDA) approval for the treatment of adults with newly diagnosed AML who are at least 75 years old or unable to tolerate intensive chemotherapy.2 However, patients with MPN-BP were excluded from the VIALE-A (ClinicalTrial.gov Identifier: NCT02993523) and VIALE-C (ClinicalTrial.gov Identifier: NCT03069352) phase 3 studies evaluating venetoclax in combination with azacitadine and low-dose cytarabine, respectively, in newly diagnosed AML.

This study included 8 patients with MPN-BP and 1 with MPN-accelerated phase (MPN-AP), defined as peripheral or bone marrow blasts of 10% to 19%, which was associated with very high-risk cytogenetics, who were treated at the Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai in New York, New York. Most of the patients in this cohort had relapsed/refractory disease, and had been treated with prior therapies.

A key study finding included the achievement of either a complete response (CR) or a CR associated with incomplete hematologic recovery (CRi) in 3 patients treated with the combination of venetoclax plus decitabine or azacitidine. In addition, stable disease as best response was achieved by 2 additional patients who received this treatment.

Of note, 2 of the 3 patients who achieved a CR/CRi had experienced disease relapse on prior HMA therapy.

This suggests a synergy with the combination that is not precluded by prior HMA exposure, the study authors remarked.

Perhaps more striking was the finding that when this therapeutic approach was used as a bridge to HSCT in 3 patients who achieved CR, CRi, or stable disease, all of them were alive at a median follow-up of 8.5 months compared with 4.2 months for the overall cohort.

However, high rates of grade 3 or higher bleeding and infection were observed in this patient cohort, and occurred in 5 and 7 patients, respectively.

Given the propensity for prolonged cytopenias with resultant complications, caution should be used in patients with baseline cytopenias, study authors noted.

In closing, the study authors stated, This is the largest report of venetoclax use in patients with MPN-AP/BP and suggests that this therapeutic strategy is a viable treatment option in this adverse risk group eligible for HSCT.

They further added that prospective clinical trial evaluation of combination HMA and venetoclax in MPN-BP is warranted.

Disclosures: Multiple authors declared affiliations with industry. Please refer to the original article for a full list of disclosures.

References

1. Tremblay D, Feld J, Dougherty M, et al. Venetoclax and hypomethylating agent combination therapy in acute myeloid leukemia secondary to a myeloproliferative neoplasm. Leuk Res. Published online September 22, 2020. doi:10.1016/j.leukres.2020.106456

2. U.S. Food and Drug Administration. FDA grants regular approval to venetoclax in combination for untreated acute myeloid leukemia [news release]. U.S. Food and Drug Administration; October 16, 2020. Accessed October 19, 2020. https://www.fda.gov/drugs/drug-approvals-and-databases/fda-grants-regular-approval-venetoclax-combination-untreated-acute-myeloid-leukemia

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Some Patients With AML Secondary to MPN May Benefit From Venetoclax in Combination With a Hypomethylating Agent - Oncology Nurse Advisor

Bone Marrow Stem Cells Comments Off on Some Patients With AML Secondary to MPN May Benefit From Venetoclax in Combination With a Hypomethylating Agent – Oncology Nurse Advisor | October 23rd, 2020

You may have heard of T cells, but Aleks Radovic-Moreno, Ph.D., Be Biopharmas co-founder, president and director, is betting on B cells as the future of cell therapies.

Our mission is to develop what we see as a new class of cell medicines that have a broad new pharmacology, he said of B cells potential. We think it's a big new white space that's enabled by the rich biology of these cells.

The Cambridge, Massachusetts-based company is capitalizingearly on research by scientists at the University of Washington School of Medicine. With a $52 million series A round in the bank, it'smaking a beeline for the clinic.

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Why the enthusiasm around B cells? The wayRadovic-Moreno sees it, they'rethe cellular gadget, if you will, that's really good at making large amounts of protein, and they also traffic to where you want them to go."

When we think about it from a drug development standpoint, now you have a system that can make a protein that you want in high quantities in places where you want it to be made, he added.

B cells may also be useful for targeting specific tissues and modulating microenvironments, or [talking] to the cells that are nearby, he said.

One of the biggest challenges to bringing Be Bio to fruition was making the products themselves. Theyre harder to engineer than other cell types thanksto their intrinsic biology, Radovic-Moreno said. Theyre also hard to make correctly and in large quantities, challenges the company only recently overcame.

Those two are the final two bottlenecks that were preventing B cells from being a viable stem cell therapy modality, he said.

RELATED: Q32 debuts with $46M to 'rebalance' innate and adaptive immunity

The applications of B cells include everything from autoimmune diseases to cancer and monogenic disorders, which are caused by variation in a single gene. B-cell therapy could eliminate the need for patients with monogenic disorders who are missing proteins to get biweekly four-hour infusions.

And that's not all. It couldalso eliminate the need for bone marrow transplants in these patients, as well asthe need for a pre-therapy round of chemotherapy, otherwise known as conditioning. For cancer patients who need conditioningahead of a stem cell treatment, the regimencan be deadly up to 10% of the time.

That's extraordinary if you think about a therapy killing patients 10% of the time, Radovic-Moreno said.

Beyond pushing Be'spipeline toward the clinic, the new fundingfrom Atlas Venture, RA Capital Management, Alta Partners, Longwood Fund and other investorswill bankroll potential partnerships and build out the company's team.

The most important thing is to build a great company, hire the best people. We want to be the best B-cell engineers in the world and in history, Radovic-Moreno said. We want to fully capitalize on the timing of this, given that it's a very kind of unusual place to be in this time and age of biotech, where you're sitting right in front of this massive blue wave, big blue ocean of possibilities so big.

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Be Biopharma debuts with $52M to advance engineered B-cell therapies - FierceBiotech

Bone Marrow Stem Cells Comments Off on Be Biopharma debuts with $52M to advance engineered B-cell therapies – FierceBiotech | October 23rd, 2020

The globalRheumatoid Arthritis Stem Cell Therapy marketstudy presents an all in all compilation of the historical, current and future outlook of the market as well as the factors responsible for such a growth. With SWOT analysis, the business study highlights the strengths, weaknesses, opportunities and threats of each Rheumatoid Arthritis Stem Cell Therapy market player in a comprehensive way. Further, the Rheumatoid Arthritis Stem Cell Therapy market report emphasizes the adoption pattern of the Rheumatoid Arthritis Stem Cell Therapy across various industries.Request Sample Reporthttps://www.factmr.com/connectus/sample?flag=S&rep_id=1001The Rheumatoid Arthritis Stem Cell Therapy market report highlights the following players:The global market for rheumatoid arthritis stem cell therapy is highly fragmented. Examples of some of the key players operating in the global rheumatoid arthritis stem cell therapy market include Mesoblast Ltd., Roslin Cells, Regeneus Ltd, ReNeuron Group plc, International Stem Cell Corporation, TiGenix and others.

The Rheumatoid Arthritis Stem Cell Therapy market report examines the operating pattern of each player new product launches, partnerships, and acquisitions has been examined in detail.Important regions covered in the Rheumatoid Arthritis Stem Cell Therapy market report include:

North America (U.S., Canada)Latin America (Mexico, Brazil)Western Europe (Germany, Italy, U.K., Spain, France, Nordic countries, BENELUX)Eastern Europe (Russia, Poland, Rest Of Eastern Europe)Asia Pacific Excluding Japan (China, India, Australia & New Zealand)JapanMiddle East and Africa (GCC, S. Africa, Rest Of MEA)

The Rheumatoid Arthritis Stem Cell Therapy market report takes into consideration the following segments by treatment type:

Allogeneic Mesenchymal stem cellsBone marrow TransplantAdipose Tissue Stem Cells

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Changing consumption pattern among individuals globally.Historical and future progress of the global Rheumatoid Arthritis Stem Cell Therapy market.Region-wise and country-wise segmentation of the Rheumatoid Arthritis Stem Cell Therapy market to understand the revenue, and growth lookout in these areas.Accurate Year-on-Year growth of the global Rheumatoid Arthritis Stem Cell Therapy market.Important trends, including proprietary technologies, ecological conservation, and globalization affecting the global Rheumatoid Arthritis Stem Cell Therapy market.

The Rheumatoid Arthritis Stem Cell Therapy market report answers important questions which include:

Which regulatory authorities have granted approval to the application of Rheumatoid Arthritis Stem Cell Therapy in Health industry?How will the global Rheumatoid Arthritis Stem Cell Therapy market grow over the forecast period?Which end use industry is set to become the leading consumer of Rheumatoid Arthritis Stem Cell Therapy by 2028?What manufacturing techniques are involved in the production of the Rheumatoid Arthritis Stem Cell Therapy?Which regions are the Rheumatoid Arthritis Stem Cell Therapy market players targeting to channelize their production portfolio?Get Full Access of the Report @https://www.factmr.com/report/1001/rheumatoid-arthritis-stem-cell-therapy-market

Pertinent aspects this study on the Rheumatoid Arthritis Stem Cell Therapy market tries to answer exhaustively are:

What is the forecast size (revenue/volumes) of the most lucrative regional market? What is the share of the dominant product/technology segment in the Rheumatoid Arthritis Stem Cell Therapy market? What regions are likely to witness sizable investments in research and development funding? What are Covid 19 implication on Rheumatoid Arthritis Stem Cell Therapy market and learn how businesses can respond, manage and mitigate the risks? Which countries will be the next destination for industry leaders in order to tap new revenue streams? Which new regulations might cause disruption in industry sentiments in near future? Which is the share of the dominant end user? Which region is expected to rise at the most dominant growth rate? Which technologies will have massive impact of new avenues in the Rheumatoid Arthritis Stem Cell Therapy market? Which key end-use industry trends are expected to shape the growth prospects of the Rheumatoid Arthritis Stem Cell Therapy market? What factors will promote new entrants in the Rheumatoid Arthritis Stem Cell Therapy market? What is the degree of fragmentation in the Rheumatoid Arthritis Stem Cell Therapy market, and will it increase in coming years?Why Choose Fact.MR?

Fact.MR follows a multi- disciplinary approach to extract information about various industries. Our analysts perform thorough primary and secondary research to gather data associated with the market. With modern industrial and digitalization tools, we provide avant-garde business ideas to our clients. We address clients living in across parts of the world with our 24/7 service availability.

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Potential impact of Covid-19 on Rheumatoid Arthritis Stem Cell Therapy Market Growth and Demand, Concludes Fact.MR - The Cloud Tribune

Bone Marrow Stem Cells Comments Off on Potential impact of Covid-19 on Rheumatoid Arthritis Stem Cell Therapy Market Growth and Demand, Concludes Fact.MR – The Cloud Tribune | October 23rd, 2020

NEW YORK, Oct. 21, 2020 (GLOBE NEWSWIRE) -- Bragar Eagel & Squire, P.C., a nationally recognized shareholder rights law firm, reminds investors that class actions have been commenced on behalf of stockholders of Mesoblast Limited (NASDAQ: MESO), Loop Industries, Inc. (NASDAQ: LOOP), Turquoise Hill Resources Ltd. (NYSE: TRQ), and Reata Pharmaceuticals, Inc. (NASDAQ: RETA). Stockholders have until the deadlines below to petition the court to serve as lead plaintiff. Additional information about each case can be found at the link provided.

Mesoblast Limited (NASDAQ: MESO)

Class Period: April 16, 2019 to October 1, 2020

Lead Plaintiff Deadline: December 7, 2020

Mesoblast develops allogeneic cellular medicines using its proprietary mesenchymal lineage cell therapy platform. Its lead product candidate, RYONCIL (remestemcel-L), is an investigational therapy comprising mesenchymal stem cells derived from bone marrow. In February 2018, the Company announced that remestemcel-L met its primary endpoint in a Phase 3 trial to treat children with steroid refractory acute graft versus host disease (aGVHD).

In early 2020, Mesoblast completed its rolling submission of its Biologics License Application (BLA) with the FDA to secure marketing authorization to commercialize remestemcel-L for children with steroid refractory aGVHD.

On August 11, 2020, the FDA released briefing materials for its Oncologic Drugs Advisory Committee (ODAC) meeting to be held on August 13, 2020. Therein, the FDA stated that Mesoblast provided post hoc analyses of other studies to further establish the appropriateness of 45% as the null Day-28 ORR for its primary endpoint. The briefing materials stated that, due to design differences between these historical studies and Mesoblasts submitted study, it is unclear that these study results are relevant to the proposed indication.

On this news, the Companys share price fell $6.09, or approximately 35%, to close at $11.33 per share on August 11, 2020.

On October 1, 2020, Mesoblast disclosed that it had received a Complete Response Letter (CRL) from the FDA regarding its marketing application for remestemcel-L for treatment of SR-aGVHD in pediatric patients. According to the CRL, the FDA recommended that the Company conduct at least one additional randomized, controlled study in adults and/or children to provide further evidence of the effectiveness of remestemcel-L for SR-aGVHD. The CRL also identified a need for further scientific rationale to demonstrate the relationship of potency measurements to the products biologic activity.

On this news, the Companys share price fell $6.56, or 35%, to close at $12.03 per share on October 2, 2020.

The complaint, filed on October 8, 2020, alleges that throughout the Class Period defendants made materially false and/or misleading statements, as well as failed to disclose material adverse facts about the Companys business, operations, and prospects. Specifically, defendants failed to disclose to investors: (1) that comparative analyses between Mesoblasts Phase 3 trial and three historical studies did not support the effectiveness of remestemcel-L for steroid refractory aGVHD due to design differences between the four studies; (2) that, as a result, the FDA was reasonably likely to require further clinical studies; (3) that, as a result, the commercialization of remestemcel-L in the U.S. was likely to be delayed; and (4) that, as a result of the foregoing, defendants positive statements about the Companys business, operations, and prospects were materially misleading and/or lacked a reasonable basis.

For more information on the Mesoblast class action go to: https://bespc.com/MESO

Loop Industries, Inc. (NASDAQ: LOOP)

Class Period: September 24, 2018 to October 12, 2020

Lead Plaintiff Deadline: December 14, 2020

On October 13, 2020, Hindenburg Research published a report alleging, among other things, that Loops scientists, under pressure from CEO Daniel Solomita, were tacitly encouraged to lie about the results of the companys process internally. The report also stated that Loops previous claims of breaking PET down to its base chemicals at a recovery rate of 100% were technically and industrially impossible, according to a former employee. Moreover, the report alleged that Executives from a division of key partner Thyssenkrupp, who Loop entered into a global alliance agreement with in December 2018, told us their partnership is on indefinite hold and that Loop underestimated both costs and complexities of its process.

On this news, the Companys share price fell $3.78, or over 32%, to close at $7.83 per share on October 13, 2020.

The complaint, filed on October 13, 2020, alleges that throughout the Class Period defendants made materially false and/or misleading statements, as well as failed to disclose material adverse facts about the Companys business, operations, and prospects. Specifically, defendants failed to disclose to investors: (1) that Loop scientists were encouraged to misrepresent the results of Loops purportedly proprietary process; (2) that Loop did not have the technology to break PET down to its base chemicals at a recovery rate of 100%; (3) that, as a result, the Company was unlikely to realize the purported benefits of Loops announced partnerships with Indorama and Thyssenkrupp; and (4) that, as a result of the foregoing, defendants positive statements about the Companys business, operations, and prospects were materially misleading and/or lacked a reasonable basis.

For more information on the Loop class action go to: https://bespc.com/Loop

Turquoise Hill Resources Ltd. (NYSE: TRQ)

Class Period: July 17, 2018 to July 31, 2019

Lead Plaintiff Deadline: December 14, 2020

Turquoise Hill is an international mining company focused on the operation and development of the Oyu Tolgoi copper-gold mine in Southern Mongolia (Oyu Tolgoi), which is the Companys principal and only material resource property. Turquoise Hills subsidiary, Oyu Tolgoi LLC, holds a 66% interest in Oyu Tolgoi, and the remainder is held by the Government of Mongolia.

Rio Tinto plc and Rio Tinto Limited are operated and managed together as single economic unit and engage in mining and metals operations in approximately 35 countries. Through their subsidiaries, Rio Tinto owns 50.8% of Turquoise Hill. A Rio Tinto subsidiary, Rio Tinto International Holdings, Inc. (Rio Tinto International or RTIH; and collectively with Rio Tinto plc and Rio Tinto Limited, Rio Tinto), is also the manager of the Oyu Tolgoi project, including having responsibility for its development and construction.

On July 31, 2019, Turquoise Hill issued a press release and Management Discussion & Analysis (MD&A) making further disclosures about the status of the project, including that Turquoise Hill took a $600 million impairment charge and a substantial deferred income tax recognition adjustment tied to the Oyu Tolgoi project, and that it suffered a loss in the second quarter. The next day, before the market open, Rio Tinto issued a release concerning in part the project status, including that it had also taken an impairment charge related to the Oyu Tolgoi project, of $800 million.

Following this news, on August 1, 2019, Turquoise Hills common stock price closed at $0.53 per share, down 8.62% from the prior days closing price of $0.58 per share.

The complaint, filed on October 15, 2020, alleges that throughout the Class Period defendants made materially false and misleading statements and omitted to disclose material facts regarding the Companys business and operations. Specifically, defendants made false and or misleading statements and/or failed to disclose that: (i) the progress of underground development of Oyu Tolgoi was not proceeding as planned; (ii) there were significant undisclosed underground stability issues that called into question the design of the mine, the projected cost and timing of production; (iii) the Companys publicly disclosed estimates of the cost, date of completion and dates for production from the underground mine were not achievable; (iv) the development capital required for the underground development of Oyu Tolgoi would cost substantially more than a billion dollars over what the Company had represented; and (v) Turquoise Hill would require additional financing and/or equity to complete the project.

For more information on the Turquoise Hill class action go to: https://bespc.com/TRQ

Reata Pharmaceuticals, Inc. (NASDAQ: RETA)

Class Period: October 15, 2019 to August 7, 2020

Lead Plaintiff Deadline: December 14, 2020

Reata is a clinical stage biopharmaceutical company that develops novel therapeutics for patients with serious or life-threatening diseases by targeting molecular pathways that regulate cellular metabolism and inflammation.

Among Reatas drug candidates under development is omaveloxolone, which is in Phase 2 clinical development to treat Friedreich's ataxia (FA). Following the announcement of positive data from the MOXIe Part 2 study of omaveloxolone for FA inOctober 2019, the Company represented that it would seek submission for marketing approval of omaveloxolone for the treatment of FA in the U.S. with the U.S. Food and Drug Administration (FDA).

OnAugust 10, 2020, Reata issued a press release announcing its second quarter 2020 financial results, wherein it disclosed that the FDA is not convinced that the MOXIe Part 2 results of the Company's study assessing omaveloxolone for the treatment of FA will support a single study approval without additional evidence that lends persuasiveness to the results, and that, [i]n preliminary comments for [a] meeting, the FDA stated that [Defendants] will need to conduct a second pivotal trial that confirms the mFARS [modified Friedreich's Ataxia Rating Scale] results of the MOXIe Part 2 study with a similar magnitude of effect.

On this news, Reatas stock price fell$51.79per share, or 33.16%, to close at$104.41per share onAugust 10, 2020.

The Complaint, filed on October 15, 2020, alleges that throughout the Class Period defendants made materially false and misleading statements regarding the Companys business. Specifically, defendants made false and/or misleading statements and/or failed to disclose that: (i) the MOXIe Part 2 study results were insufficient to support a single study marketing approval of omaveloxolone for the treatment of FA in the U.S. without additional evidence; (ii) as a result, it was foreseeable that the FDA would not accept marketing approval of omaveloxolone for the treatment of FA in the U.S. based on the MOXIe Part 2 study results; and (iii) as a result, the Company's public statements were materially false and misleading at all relevant times.

For more information on the Reata class action go to: https://bespc.com/REATA

About Bragar Eagel & Squire, P.C.:Bragar Eagel & Squire, P.C. is a nationally recognized law firm with offices in New York and California. The firm represents individual and institutional investors in commercial, securities, derivative, and other complex litigation in state and federal courts across the country. For more information about the firm, please visit http://www.bespc.com. Attorney advertising. Prior results do not guarantee similar outcomes.

Contact Information:Bragar Eagel & Squire, P.C.Brandon Walker, Esq. Melissa Fortunato, Esq.Marion Passmore, Esq.(212) 355-4648investigations@bespc.comwww.bespc.com

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Bragar Eagel & Squire, PC Reminds Investors That Class Action Lawsuits Have Been Filed Against Mesoblast, Loop Industries, Turquoise Hill...

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Company Announcement

Copenhagen, Denmark; October 21, 2020 Genmab A/S (Nasdaq: GMAB) announced today positive topline results from the second part of the Phase 3 CASSIOPEIA (MMY3006) study of daratumumab monotherapy as maintenance treatment versus observation (no treatment) for patients with newly diagnosed multiple myeloma eligible for autologous stem cell transplant (ASCT). The second part of the study, which is being conducted by the French Intergroupe Francophone du Myelome (IFM) in collaboration with the Dutch-Belgian Cooperative Trial Group for Hematology Oncology (HOVON) and Janssen Research & Development, LLC (Janssen), met the primary endpoint of improving progression free survival (PFS) at a pre-planned interim analysis (Hazard Ratio (HR) = 0.53 (95% CI 0.42 0.68), p < 0.0001) resulting in a 47% reduction in the risk of progression or death in patients treated with daratumumab. The safety profile observed in this study was consistent with the known safety profile of daratumumab and no new safety signals were observed.

Based on the results at the pre-planned interim analysis conducted by an Independent Data Monitoring Committee (IDMC), it was recommended to unblind the study results. Janssen Biotech, Inc., which licensed daratumumab from Genmab in 2012, plans to discuss the potential for a regulatory submission for this indication with health authorities, and plans to submit the data to an upcoming medical conference and for publication in a peer-reviewed journal.

Following the positive data from the first part of the CASSIOPEIA study, we are very pleased to see this benefit. We are appreciative of the efforts of the IFM, of HOVON and of Janssen for their work on this study, said Jan van de Winkel, Ph.D., Chief Executive Officer of Genmab.

About the CASSIOPEIA (MMY3006) StudyThis Phase 3 study is a randomized, open-label, multicenter study, conducted by the IFM in collaboration with the HOVON and Janssen, which includes 1,085 newly diagnosed subjects with previously untreated symptomatic multiple myeloma who were eligible for high dose chemotherapy and ASCT. In the first part of the study, patients were randomized to receive induction and consolidation treatment with daratumumab combined with bortezomib, thalidomide and dexamethasone (VTd) or VTd alone. The primary endpoint was the number of patients that achieved a stringent complete response (sCR). In the second part of the study, patients that achieved a response underwent a second randomization to either receive maintenance treatment of daratumumab 16 mg/kg every 8 weeks for up to 2 years versus no further treatment (observation). The primary endpoint of this part of the study is progression free survival.

About Multiple MyelomaMultiple myeloma is an incurable blood cancer that starts in the bone marrow and is characterized by an excess proliferation of plasma cells.1 Multiple myeloma is the third most common blood cancer in the U.S., after leukemia and lymphoma.2 Approximately 26,000 new patients were expected to be diagnosed with multiple myeloma and approximately 13,650 people were expected to die from the disease in the U.S. in 2018.3 Globally, it was estimated that 160,000 people were diagnosed and 106,000 died from the disease in 2018.4 While some patients with multiple myeloma have no symptoms at all, most patients are diagnosed due to symptoms which can include bone problems, low blood counts, calcium elevation, kidney problems or infections.5

About DARZALEX (daratumumab)DARZALEX (daratumumab) has become a backbone therapy in the treatment of multiple myeloma. DARZALEX intravenous infusion is indicated for the treatment of adult patients in the United States: in combination with carfilzomib and dexamethasone for the treatment of patients with relapsed/refractory multiple myeloma who have received one to three previous lines of therapy; in combination with bortezomib, thalidomide and dexamethasone as treatment for patients newly diagnosed with multiple myeloma who are eligible for autologous stem cell transplant; in combination with lenalidomide and dexamethasone for the treatment of patients with newly diagnosed multiple myeloma who are ineligible for autologous stem cell transplant; in combination with bortezomib, melphalan and prednisone for the treatment of patients with newly diagnosed multiple myeloma who are ineligible for autologous stem cell transplant; in combination with lenalidomide and dexamethasone, or bortezomib and dexamethasone, for the treatment of patients with multiple myeloma who have received at least one prior therapy; in combination with pomalidomide and dexamethasone for the treatment of patients with multiple myeloma who have received at least two prior therapies, including lenalidomide and a proteasome inhibitor (PI); and as a monotherapy for the treatment of patients with multiple myeloma who have received at least three prior lines of therapy, including a PI and an immunomodulatory agent, or who are double-refractory to a PI and an immunomodulatory agent.6 DARZALEX is the first monoclonal antibody (mAb) to receive U.S. Food and Drug Administration (U.S. FDA) approval to treat multiple myeloma.

DARZALEX is indicated for the treatment of adult patients in Europe via intravenous infusion or subcutaneous administration: in combination with bortezomib, thalidomide and dexamethasone as treatment for patients newly diagnosed with multiple myeloma who are eligible for autologous stem cell transplant; in combination with lenalidomide and dexamethasone for the treatment of patients with newly diagnosed multiple myeloma who are ineligible for autologous stem cell transplant; in combination with bortezomib, melphalan and prednisone for the treatment of adult patients with newly diagnosed multiple myeloma who are ineligible for autologous stem cell transplant; for use in combination with lenalidomide and dexamethasone, or bortezomib and dexamethasone, for the treatment of adult patients with multiple myeloma who have received at least one prior therapy; and as monotherapy for the treatment of adult patients with relapsed and refractory multiple myeloma, whose prior therapy included a PI and an immunomodulatory agent and who have demonstrated disease progression on the last therapy7. Daratumumab is the first subcutaneous CD38 antibody approved in Europe for the treatment of multiple myeloma. The option to split the first infusion of DARZALEX over two consecutive days has been approved in both Europe and the U.S.

In Japan, DARZALEX intravenous infusion is approved for the treatment of adult patients: in combination with lenalidomide and dexamethasone for the treatment of patients with newly diagnosed multiple myeloma who are ineligible for autologous stem cell transplant; in combination with bortezomib, melphalan and prednisone for the treatment of patients with newly diagnosed multiple myeloma who are ineligible for autologous stem cell transplant; in combination with lenalidomide and dexamethasone, or bortezomib and dexamethasone for the treatment of relapsed or refractory multiple myeloma. DARZALEX is the first human CD38 monoclonal antibody to reach the market in the United States, Europe and Japan. For more information, visit http://www.DARZALEX.com.

DARZALEX FASPRO (daratumumab and hyaluronidase-fihj), a subcutaneous formulation of daratumumab, is approved in the United States for the treatment of adult patients with multiple myeloma: in combination with bortezomib, melphalan and prednisone in newly diagnosed patients who are ineligible for ASCT; in combination with lenalidomide and dexamethasone in newly diagnosed patients who are ineligible for ASCT and in patients with relapsed or refractory multiple myeloma who have received at least one prior therapy; in combination with bortezomib and dexamethasone in patients who have received at least one prior therapy; and as monotherapy, in patients who have received at least three prior lines of therapy including a PI and an immunomodulatory agent or who are double-refractory to a PI and an immunomodulatory agent.8 DARZALEX FASPRO is the first subcutaneous CD38 antibody approved in the U.S. for the treatment of multiple myeloma.

Daratumumab is a human IgG1k monoclonal antibody (mAb) that binds with high affinity to the CD38 molecule, which is highly expressed on the surface of multiple myeloma cells. Daratumumab triggers a persons own immune system to attack the cancer cells, resulting in rapid tumor cell death through multiple immune-mediated mechanisms of action and through immunomodulatory effects, in addition to direct tumor cell death, via apoptosis (programmed cell death).6,9,10,11,12

Daratumumab is being developed by Janssen Biotech, Inc. under an exclusive worldwide license to develop, manufacture and commercialize daratumumab from Genmab. A comprehensive clinical development program for daratumumab is ongoing, including multiple Phase 3 studies in smoldering, relapsed and refractory and frontline multiple myeloma settings. Additional studies are ongoing or planned to assess the potential of daratumumab in other malignant and pre-malignant diseases in which CD38 is expressed, such as amyloidosis and T-cell acute lymphocytic leukemia (ALL). Daratumumab has received two Breakthrough Therapy Designations from the U.S. FDA for certain indications of multiple myeloma, including as a monotherapy for heavily pretreated multiple myeloma and in combination with certain other therapies for second-line treatment of multiple myeloma.

About Genmab Genmab is a publicly traded, international biotechnology company specializing in the creation and development of differentiated antibody therapeutics for the treatment of cancer. Founded in 1999, the company is the creator of the following approved antibodies: DARZALEX (daratumumab, under agreement with Janssen Biotech, Inc.) for the treatment of certain multiple myeloma indications in territories including the U.S., Europe and Japan, Kesimpta (subcutaneous ofatumumab, under agreement with Novartis AG), for the treatment of adults with relapsing forms of multiple sclerosis in the U.S. and TEPEZZA (teprotumumab, under agreement with Roche granting sublicense to Horizon Therapeutics plc) for the treatment of thyroid eye disease in the U.S. A subcutaneous formulation of daratumumab, known as DARZALEX FASPRO (daratumumab and hyaluronidase-fihj) in the U.S., has been approved in the U.S. and Europe for the treatment of adult patients with certain multiple myeloma indications. The first approved Genmab created therapy, Arzerra (ofatumumab, under agreement with Novartis AG), approved for the treatment of certain chronic lymphocytic leukemia indications, is available in Japan and is also available in other territories via compassionate use or oncology access programs. Daratumumab is in clinical development by Janssen for the treatment of additional multiple myeloma indications, other blood cancers and amyloidosis. Genmab also has a broad clinical and pre-clinical product pipeline. Genmab's technology base consists of validated and proprietary next generation antibody technologies - the DuoBody platform for generation of bispecific antibodies, the HexaBody platform, which creates effector function enhanced antibodies, the HexElect platform, which combines two co-dependently acting HexaBody molecules to introduce selectivity while maximizing therapeutic potency and the DuoHexaBody platform, which enhances the potential potency of bispecific antibodies through hexamerization. The company intends to leverage these technologies to create opportunities for full or co-ownership of future products. Genmab has alliances with top tier pharmaceutical and biotechnology companies. Genmab is headquartered in Copenhagen, Denmark with sites in Utrecht, the Netherlands, Princeton, New Jersey, U.S. and Tokyo, Japan.

Contact: Marisol Peron, Corporate Vice President, Communications & Investor Relations T: +1 609 524 0065; E: mmp@genmab.com

For Investor Relations: Andrew Carlsen, Senior Director, Investor RelationsT: +45 3377 9558; E: acn@genmab.com

This Company Announcement contains forward looking statements. The words believe, expect, anticipate, intend and plan and similar expressions identify forward looking statements. Actual results or performance may differ materially from any future results or performance expressed or implied by such statements. The important factors that could cause our actual results or performance to differ materially include, among others, risks associated with pre-clinical and clinical development of products, uncertainties related to the outcome and conduct of clinical trials including unforeseen safety issues, uncertainties related to product manufacturing, the lack of market acceptance of our products, our inability to manage growth, the competitive environment in relation to our business area and markets, our inability to attract and retain suitably qualified personnel, the unenforceability or lack of protection of our patents and proprietary rights, our relationships with affiliated entities, changes and developments in technology which may render our products or technologies obsolete, and other factors. For a further discussion of these risks, please refer to the risk management sections in Genmabs most recent financial reports, which are available on http://www.genmab.com and the risk factors included in Genmabs most recent Annual Report on Form 20-F and other filings with the U.S. Securities and Exchange Commission (SEC), which are available at http://www.sec.gov. Genmab does not undertake any obligation to update or revise forward looking statements in this Company Announcement nor to confirm such statements to reflect subsequent events or circumstances after the date made or in relation to actual results, unless required by law.

Genmab A/S and/or its subsidiaries own the following trademarks: Genmab; the Y-shaped Genmab logo; Genmab in combination with the Y-shaped Genmab logo; HuMax; DuoBody; DuoBody in combination with the DuoBody logo; HexaBody; HexaBody in combination with the HexaBody logo; DuoHexaBody; HexElect; and UniBody. Arzerra and Kesimpta are trademarks of Novartis AG or its affiliates. DARZALEX and DARZALEX FASPRO are trademarks of Janssen Pharmaceutica NV. TEPEZZA is a trademark of Horizon Therapeutics plc.

1 American Cancer Society. "Multiple Myeloma Overview." Available at http://www.cancer.org/cancer/multiplemyeloma/detailedguide/multiple-myeloma-what-is-multiple-myeloma.Accessed June 2016.2 National Cancer Institute. "A Snapshot of Myeloma." Available at http://www.cancer.gov/research/progress/snapshots/myeloma. Accessed June 2016. 3 Globocan 2018. United States of America Fact Sheet. Available at http://gco.iarc.fr/today/data/factsheets/840-united-states-of-america-fact-sheets.pdf.4 Globocan 2018. World Fact Sheet. Available at http://gco.iarc.fr/today/data/factsheets/populations/900-world-fact-sheets.pdf. Accessed December 2018.5 American Cancer Society. "How is Multiple Myeloma Diagnosed?" http://www.cancer.org/cancer/multiplemyeloma/detailedguide/multiple-myeloma-diagnosis. Accessed June 20166 DARZALEX Prescribing information, August 2020 https://www.accessdata.fda.gov/drugsatfda_docs/label/2020/761036s029lbl.pdf Last accessed August 20207 DARZALEX Summary of Product Characteristics, available at https://www.ema.europa.eu/en/medicines/human/EPAR/darzalex Last accessed June 20208 DARZALEX FASPRO Prescribing information, May 2020. Available at: https://www.accessdata.fda.gov/drugsatfda_docs/label/2020/761145s000lbl.pdf Last accessed May 20209 De Weers, M et al. Daratumumab, a Novel Therapeutic Human CD38 Monoclonal Antibody, Induces Killing of Multiple Myeloma and Other Hematological Tumors. The Journal of Immunology. 2011; 186: 1840-1848.10 Overdijk, MB, et al. Antibody-mediated phagocytosis contributes to the anti-tumor activity of the therapeutic antibody daratumumab in lymphoma and multiple myeloma. MAbs. 2015; 7: 311-21.11 Krejcik, MD et al. Daratumumab Depletes CD38+ Immune-regulatory Cells, Promotes T-cell Expansion, and Skews T-cell Repertoire in Multiple Myeloma. Blood. 2016; 128: 384-94.12 Jansen, JH et al. Daratumumab, a human CD38 antibody induces apoptosis of myeloma tumor cells via Fc receptor-mediated crosslinking.Blood. 2012; 120(21): abstract 2974.

Company Announcement no. 45CVR no. 2102 3884LEI Code 529900MTJPDPE4MHJ122

Genmab A/SKalvebod Brygge 431560 Copenhagen VDenmark

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Genmab Announces IFM, HOVON and Janssen Achieve Positive Topline Results in Second Part of Phase 3 CASSIOPEIA Study of Daratumumab in Multiple Myeloma...

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The family of seven known human coronaviruses are known for their impact on the respiratory tract, not the heart. However, the most recent coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has marked tropism for the heart and can lead to myocarditis (inflammation of the heart), necrosis of its cells, mimicking of a heart attack, arrhythmias, and acute or protracted heart failure (muscle dysfunction). These complications, which at times are the only features of coronavirus disease 2019 (COVID-19) clinical presentation, have occurred even in cases with mild symptoms and in people who did not experience any symptoms. Recent findings of heart involvement in young athletes, including sudden death, have raised concerns about the current limits of our knowledge and potentially high risk and occult prevalence of COVID-19 heart manifestations.

The four common cold human coronavirusesHCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1have not been associated with heart abnormalities. There were isolated reports of patients with Middle East respiratory syndrome (MERS; caused by MERS-CoV) with myocarditis and a limited number of case series of cardiac disease in patients with SARS (caused by SARS-CoV) (1). Therefore, a distinct feature of SARS-CoV-2 is its more extensive cardiac involvement, which may also be a consequence of the pandemic and the exposure of tens of millions of people to the virus.

What appears to structurally differentiate SARS-CoV-2 from SARS is a furin polybasic site that, when cleaved, broadens the types of cells (tropism) that the virus can infect (2). The virus targets the angiotensin-converting enzyme 2 (ACE2) receptor throughout the body, facilitating cell entry by way of its spike protein, along with the cooperation of the cellular serine protease transmembrane protease serine 2 (TMPRSS2), heparan sulfate, and other proteases (3). The heart is one of the many organs with high expression of ACE2. Moreover, the affinity of SARS-CoV-2 to ACE2 is significantly greater than that of SARS (4). The tropism to other organs beyond the lungs has been studied from autopsy specimens: SARS-CoV-2 genomic RNA was highest in the lungs, but the heart, kidney, and liver also showed substantial amounts, and copies of the virus were detected in the heart from 16 of 22 patients who died (5). In an autopsy series of 39 patients dying from COVID-19, the virus was not detectable in the myocardium in 38% of patients, whereas 31% had a high viral load above 1000 copies in the heart (6).

Accordingly, SARS-CoV-2 infection can damage the heart both directly and indirectly (see the figure). SARS-CoV-2 exhibited a striking ability to infect cardiomyocytes derived from induced pluripotent stem cells (iPSCs) in vitro, leading to a distinctive pattern of heart muscle cell fragmentation, with complete dissolution of the contractile machinery (7). Some of these findings were verified from patient autopsy specimens. In another iPSC study, SARS-CoV-2 infection led to apoptosis and cessation of beating within 72 hours of exposure (8). Besides directly infecting heart muscle cells, viral entry has been documented in the endothelial cells that line the blood vessels to the heart and multiple vascular beds. A secondary immune response to the infected heart and endothelial cells (endothelitis) is just one dimension of many potential indirect effects. These include dysregulation of the renin-angiotensin-aldosterone system that modulates blood pressure, and activation of a proinflammatory response involving platelets, neutrophils, macrophages, and lymphocytes, with release of cytokines and a prothrombotic state. A propensity for clotting, both in the microvasculature and large vessels, has been reported in multiple autopsy series and in young COVID-19 patients with strokes.

There is a diverse spectrum of cardiovascular manifestations, ranging from limited necrosis of heart cells (causing injury), to myocarditis, to cardiogenic shock (an often fatal inability to pump sufficient blood). Cardiac injury, as reflected by concentrations of troponin (a cardiac musclespecific enzyme) in the blood, is common with COVID-19, occurring in at least one in five hospitalized patients and more than half of those with preexisting heart conditions. Such myocardial injury is a risk factor for in-hospital mortality, and troponin concentration correlates with risk of mortality. Furthermore, patients with higher troponin amounts have markers of increased inflammation [including C-reactive protein, interleukin-6 (IL-6), ferritin, lactate dehydrogenase (LDH), and high neutrophil count] and heart dysfunction (amino-terminal pro-Btype natriuretic peptide) (9).

More worrisome than the pattern of limited injury is myocarditis: diffuse inflammation of the heart, usually representing a variable admixture of injury and the inflammatory response to the injury that can extend throughout the three layers of the human heart to the pericardium (which surrounds the heart). Unlike SARS-associated myocarditis, which did not exhibit lymphocyte infiltration, this immune and inflammatory response is a typical finding at autopsy after SARS-CoV-2 infections. Involvement of myocytes, which orchestrate electrical conduction, can result in conduction block and malignant ventricular arrhythmias, both of which can lead to cardiac arrest.

Along with such in-hospital arrythmias, there have been reports of increased out-of-hospital cardiac arrest and sudden death in multiple geographic regions of high COVID-19 spread, such as the 77% increase in Lombardy, Italy, compared with the prior year (10). There have been many reports of myocarditis simulating a heart attack, owing to the cluster of chest pain symptoms, an abnormal electrocardiogram, and increased cardiac-specific enzymes in the blood, even in patients as young as a 16-year-old boy. When there is extensive and diffuse heart muscle damage, heart failure, acute cor pulmonale (right heart failure and possible pulmonary emboli), and cardiogenic shock can occur.

COVID-19associated heart dysfunction can also be attributed to other pathways, including Takotsubo syndrome (also called stress cardiomyopathy), ischemia from endothelitis and related atherosclerotic plaque rupture with thrombosis, and the multisystem inflammatory syndrome of children (MIS-C). The underlying mechanism of stress cardiomyopathy is poorly understood but has markedly increased during the pandemic. MIS-C is thought to be immune-mediated and manifests with a spectrum of cardiovascular features, including vasculitis, coronary artery aneurysms, and cardiogenic shock. This syndrome is not exclusive to children because the same clinical features have been the subject of case reports in adults, such as in a 45-year-old man (11).

Recent series of COVID-19 patients undergoing magnetic resonance imaging (MRI) or echocardiography of the heart have provided some new insights about cardiac involvement (1214). In a cohort of 100 patients recovered from COVID-19, 78 had cardiac abnormalities, including 12 of 18 patients without any symptoms, and 60 had ongoing myocardial inflammation, which is consistent with myocarditis (12). The majority of more than 1200 patients in a large prospective cohort with COVID-19 had echocardiographic abnormalities (13). This raises concerns about whether there is far more prevalent heart involvement than has been anticipated, especially because at least 30 to 40% of SARS-CoV-2 infections occur without symptoms. Such individuals may have underlying cardiac pathology.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has the potential to directly and indirectly induce cardiac damage.

To date, there have been four small series of asymptomatic individuals with bona fide infections who underwent chest computed tomography (CT) scans to determine whether there were lung abnormalities consistent with COVID-19. Indeed, half of the asymptomatic people showed lung CT features that were seen in patients with symptoms. But so far, there have been minimal cardiac imaging studies in people who test positive for SARS-CoV-2 or are seropositive but without symptoms. Furthermore, the time course of resolution or persistence of any organ abnormalities after SARS-CoV-2 infection has not yet been reported. With a high proportion of silent infections despite concurrent evidence of internal organ damage, there is a fundamental and large hole in our knowledge base.

In contrast to people without symptoms, there is a substantial proportion of people who suffer a long-standing, often debilitating illness, called long-COVID. Typical symptoms include fatigue, difficulty in breathing, chest pain, and abnormal heart rhythm. An immunologic basis is likely but has yet to be determined. Nor have such patients undergone systematic cardiovascular assessment for possible myocarditis or other heart abnormalities, such as fibrosis, which could account for some of the enduring symptoms. It would not be surprising in the future for patients to present with cardiomyopathy of unknown etiology and test positive for SARS-CoV-2 antibodies. However, attributing such cardiomyopathy to the virus may be difficult given the high prevalence of infections, and ultimately a biopsy might be necessary to identify virus particles to support causality.

Cardiac involvement in athletes has further elevated the concerns. A 27-year-old professional basketball player, recovered from COVID-19, experienced sudden death during training. Several college athletes have been found to have myocarditis (14), including 4 of 26 (15%) in a prospective study from Ohio State University (15), along with one of major league baseball's top pitchers. Collectively, these young, healthy individuals had mild COVID-19 but were subsequently found to have unsuspected cardiac pathology. This same demographic groupyoung and healthyare the most common to lack symptoms after SARS-CoV-2 infections, which raises the question of how many athletes have occult cardiac disease? Systematic assessment of athletes who test positive for SARS-CoV-2, irrespective of symptoms, with suitable controls through some form of cardiac imaging and arrhythmia screening seems prudent until more is understood.

The most intriguing question that arises is why do certain individuals have a propensity for heart involvement after SARS-CoV-2 infection? Once recognized a few months into the pandemic, the expectation was that cardiac involvement would chiefly occur in patients with severe COVID-19. Clearly, it is more common than anticipated, but the true incidence is unknown. It is vital to determine what drives this pathogenesis. Whether it represents an individual's inflammatory response, an autoimmune phenomenon, or some other explanation needs to be clarified. Beyond preventing SARS-CoV-2 infections, the goal of averting cardiovascular involvement is paramount. The marked heterogeneity of COVID-19, ranging from lack of symptoms to fatality, is poorly understood. A newly emerged virus, widely circulating throughout the human population, with a panoply of disease manifestations, all too often occult, has made this especially daunting to unravel.

Acknowledgments: E.J.T. is supported by National Institutes of Health grant UL1 TR001114.

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COVID-19 can affect the heart - Science Magazine

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