The death rate of COVID-19 infection in different SARS-CoV-2 variants was related to C-reactive protein gene polymorphisms.

The serum level of C-reactive protein (CRP) is a significant independent risk factor for Coronavirus disease 2019 (COVID-19). A link was found between serum CRP and genetic diversity within the CRP gene in earlier research. This study examined whether CRP rs1205 and rs1800947 polymorphisms were associated with COVID-19 mortality among various severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) variants. We genotyped CRP rs1205 and rs1800947 polymorphisms in 2023 deceased and 2307 recovered patients using the polymerase chain reaction-restriction fragment length polymorphism method. There was a significant difference between the recovered and the deceased patients in terms of the minor allele frequency of CRP rs1205 T and rs1800947 G. In all three variants, COVID-19 mortality rates were associated with CRP rs1800947 GG genotype. Furthermore, CRP rs1205 CC and rs1800947 GG genotypes showed higher CRP levels. It was found that the G-T haplotype was prevalent in all SARS-CoV-2 variants. The C-C and C-T haplotypes were statistically significant in Delta and Omicron BA.5 variants, respectively. In conclusion, polymorphisms within the CRP gene may relate to serum CRP levels and mortality among COVID-19 patients. In order to verify the utility of CRP polymorphism correlation in predicting COVID-19 mortality, a replication of these results is needed.

Immunomodulatory potential of human clonal mesenchymal stem cells and their extracellular vesicle subpopulations in an inflammatory-mediated diabetic Rhesus monkey model.

AIMS: This study aimed to investigate the therapeutic potential of a homogenous clonal population of mesenchymal stem cells (cMSC) and their extracellular vesicles (cMSC-EV) subpopulations on isolated rat islets in vitro and in inflammatory-mediated type 1 diabetes (T1D) non-human primate models. MAIN METHODS: EV subpopulations were isolated from human bone marrow-derived cMSC supernatant by low- and high-speed ultracentrifuge (EV-20K and EV-U110K) and sucrose density gradient (EV-S110K). The EVs were characterized generally and for the level of albumin, acetylcholinesterase (AChE) activity, co-isolate apoptotic markers, and expression of CD63+/annexin V+. Rat islet-derived single cells (iSCs) proliferation was measured using a Ki-67 proliferation assay. Diabetes was induced by multiple low-dose administrations of streptozotocin in rhesus monkeys. The diabetic monkeys were divided into three groups: the cMSC group, received two injections of 1.5 × 106 cMSC/kg body weight; the EV group received two injections of EVs isolated from 1.5 × 106 cMSC/kg, and the vehicle group received phosphate-buffered saline. KEY FINDINGS: EV-S110K showed higher AChE activity, lower expression of CD63+/annexin V+, and lower apoptotic co-isolates. EV-S110K induced ß-cell proliferation in vitro in a dose-dependent manner. The administration of EV-S110K and/or cMSC in diabetic monkeys demonstrated no significant changes in general diabetic indices and ß-cell mass in the pancreas of the monkeys. Both treatments demonstrated a lowering trend in blood glucose levels and reduced pro-inflammatory cytokines. In contrast, regulatory T cells and anti-inflammatory cytokines were increased. SIGNIFICANCE: cMSC and cMSC-EV provided initial evidence to attenuate clinical symptoms in inflammatory-mediated T1D non-human primates through immunomodulation.

Homogenous subpopulation of human mesenchymal stem cells and their extracellular vesicles restore function of endometrium in an experimental rat model of Asherman syndrome.

BACKGROUND: Asherman syndrome (AS), or intrauterine adhesions, is a main cause of infertility in reproductive age women after endometrial injury. Mesenchymal stem cells (MSCs) and their extracellular vesicles (EVs) are promising candidates for therapies that repair damaged endometria. However, concerns about their efficacy are attributed to heterogeneity of the cell populations and EVs. A homogenous population of MSCs and effective EV subpopulation are needed to develop potentially promising therapeutic options in regenerative medicine. METHODS: AS model was induced by mechanical injury in adult rat uteri. Then, the animals were treated immediately with homogeneous population of human bone marrow-derived clonal MSCs (cMSCs), heterogenous parental MSCs (hMSCs), or cMSCs-derived EV subpopulations (EV20K and EV110K). The animals were sacrificed two weeks post-treatment and uterine horns were collected. The sections were taken, and hematoxylin-eosin was used to examine the repair of endometrial structure. Fibrosis was measured by Masson's trichrome staining and α-SMA and cell proliferation by Ki67 immunostaining. The function of the uteri was explored by the result of mating trial test. Expression changes of TNFα, IL-10, VEGF, and LIF were assayed by ELISA. RESULTS: Histological analysis indicated fewer glands, thinner endometria, increased fibrotic areas, and decreased proliferation of epithelial and stroma of the uteri in the treated compared with intact and sham-operated animals. However, these parameters improved after transplantation of both types of cMSCs and hMSCs and/or both cryopreserved EVs subpopulations. The cMSCs demonstrated more successful implantation of the embryos in comparison with hMSCs. The tracing of the transplanted cMSCs and EVs showed that they migrated and localized in the uteri. Protein expression analysis results demonstrated downregulation of proinflammatory factor TNFα and upregulation of anti-inflammatory cytokine IL-10, and endometrial receptivity cytokines VEGF and LIF in cMSC- and EV20K-treated animals. CONCLUSION: Transplantation of MSCs and EVs contributed to endometrial repair and restoration of reproductive function, likely by inhibition of excessive fibrosis and inflammation, enhancement of endometrial cell proliferation, and regulation of molecular markers related to endometrial receptivity. Compared to classical hMSCs, cMSCs were more efficient than hMSCs in restoration of reproductive function. Moreover, EV20K is more cost-effective and feasible for prevention of AS in comparison with conventional EVs (EV110K).

Regenerative potential of different extracellular vesicle subpopulations derived from clonal mesenchymal stem cells in a mouse model of chemotherapy-induced premature ovarian failure.

AIMS: Some studies have shown that mesenchymal stem cells (MSCs) and their derived extracellular vesicles (MSC-EVs) can restore ovarian function in premature ovarian failure (POF), however, concerns about their efficacy are attributed to the heterogeneity of the cell populations and EVs. Here, we assessed the therapeutic potential of a homogeneous population of clonal MSCs (cMSCs) and their EVs subpopulations in a mouse model of POF. MAIN METHODS: Granulosa cells were treated with cyclophosphamide (Cy) in the absence or presence of cMSCs, or cMSCs-derived EV subpopulations (EV20K and EV110K, isolated by high-speed centrifugation and differential ultracentrifugation, respectively). In addition, POF mice were treated with cMSCs, EV20K and/or EV110K. KEY FINDINGS: cMSC and both EV types protected granulosa cells from Cy-induced damage. Calcein-EVs were detected in the ovaries. Moreover, cMSC and both EV subpopulations significantly increased body weight, ovary weight, and the number of follicles, restored FSH, E2, and AMH levels, increased the granulosa cell numbers and restored the fertility of POF mice. cMSC, EV20K, and EV110K alleviated inflammatory-related genes expression (Tnf-α and IL8), and improved angiogenesis via upregulation expression of Vegf and Igf1 at the mRNA level and VEGF and αSMA at the protein level. They also inhibited apoptosis through the PI3K/AKT signaling pathway. SIGNIFICANCE: The administration of cMSCs and two cMSC-EVs subpopulations improved ovarian function and restored fertility in a POF model. EV20K is more cost-effective and feasible in terms of isolation, particularly in good manufacturing practice (GMP) facilities for treatment of POF patients in comparison with conventional EVs (EV110K).

Human umbilical cord serum as an alternative to fetal bovine serum for in vitro expansion of umbilical cord mesenchymal stromal cells.

In the use of bovine fetal serum (FBS) there is concern about the possibility of disease transmission from animal to human. Therefore, it seems necessary to create culture conditions free of animal serum, especially in cell therapy. The aim of this study was to evaluate the feasibility of replacing human umbilical cord serum (hUCS) with FBS for in vitro expansion of umbilical cord mesenchymal stromal/stem cells (UC-MSCs). Here, UC-MSCs were cultured for five days in media supplemented either by hUCS or commercial FBS (Gibco and HyClone) to compare their viability, proliferation, morphology, Immunophenotype and differentiation potential. Our data shows that use of 5% and/or 10% hUCS, resulted in a tenfold increase in the number of MSCs; While in the presence of commercial FBS, this figure reached a maximum of five times. Notably, the rate of cell proliferation in the group containing 2% hUCS was the same as the groups containing 10% commercial FBS. Furthermore, there was no significant difference between groups in terms of viability, surface markers, and multilineage differentiation potential. These results demonstrated that hUCS can efficiently replace FBS for the routine culture of MSCs and can be used ideally in manufacturing process of UC-MSCs in cell therapy industry.

Standard toxicity study of clinical-grade allogeneic human bone marrow-derived clonal mesenchymal stromal cells.

INTRODUCTION: Mesenchymal stromal cells (MSCs) have opened a new window to treat inflammatory and non-inflammatory diseases. Nonetheless, their clinical applications require rigorous control and monitoring procedures to ensure full compliance with the principles of good manufacturing practice (GMP). Various evaluations should be passed in conjunction with the development of these newly emerging therapeutic products from bench-to-bedside. These evaluations include in vitro characterization, preclinical studies, and clinical trials to ensure product safety and efficacy. Therefore, a robust and well-designed preclinical study is critical to confirm product safety. This study aims to determine the probable toxicity effects of local and systemic injections of cryopreserved human bone marrow-derived clonal MSCs (BM-cMSCs) during subacute and subchronic periods of time. METHODS: BM-cMSCs were characterized according to the International Society for Cell and Gene Therapy (ISCT) criteria for MSCs. Both safety and toxicity of the BM-cMSCs population produced under GMP-compatible conditions were assessed in both sexes of Sprague Dawley (SD) rats via systemic intravenous (IV) administration and local injection in intervertebral disc (IVD). Behavioral changes, clinical signs of toxicity, and changes in body weight, water and food consumption were the important variables for product toxicity testing over 14 consecutive days during the subacute period and 90 consecutive days during the subchronic period. At the end of the assessment periods, the rats were killed for histopathology analysis of the target tissues. The BM-cMSCs potential for tumorigenicity was checked in nude mice. RESULTS: Single IV and IVD injections of BM-cMSCs did not cause significant signs of clinical toxicity, or changes in laboratory and histopathology data during the subacute (14 day) and subchronic (90 day) periods. Ex vivo-expanded and cryopreserved BM-cMSCs did not induce tumor formation in nude mice. CONCLUSION: The results suggest that local and systemic administrations of xenogeneic BM-cMSCs in both sexes of SD rats do not cause toxicity during the subacute and subchronic periods of time. Also, BM-cMSCs were non-tumorigenic in nude mice.

A Roadmap for the Production of a GMP-Compatible Cell Bank of Allogeneic Bone Marrow-Derived Clonal Mesenchymal Stromal Cells for Cell Therapy Applications.

BACKGROUND: Allogeneic mesenchymal stromal cells (MSCs) have been used extensively in various clinical trials. Nevertheless, there are concerns about their efficacy, attributed mainly to the heterogeneity of the applied populations. Therefore, producing a consistent population of MSCs is crucial to improve their therapeutic efficacy. This study presents a good manufacturing practice (GMP)-compatible and cost-effective protocol for manufacturing, banking, and lot-release of a homogeneous population of human bone marrow-derived clonal MSCs (cMSCs). METHODS: Here, cMSCs were isolated based on the subfractionation culturing method. Afterward, isolated clones that could reproduce up to passage three were stored as the seed stock. To select proliferative clones, we used an innovative, cost-effective screening strategy based on lengthy serial passaging. Finally, the selected clones re-cultured from the seed stock to establish the following four-tired cell banking system: initial, master, working, and end of product cell banks (ICB, MCB, WCB, and EoPCB). RESULTS: Through a rigorous screening strategy, three clones were selected from a total of 21 clones that were stored during the clonal isolation process. The selected clones met the identity, quality, and safety assessments criteria. The validated clones were stored in the four-tiered cell bank system under GMP conditions, and certificates of analysis were provided for the three-individual ready-to-release batches. Finally, a stability study validated the EoPCB, release, and transport process of the frozen final products. CONCLUSION: Collectively, this study presents a technical and translational overview of a GMP-compatible cMSCs manufacturing technology that could lead to the development of similar products for potential therapeutic applications.

Improved Differentiation of hESC-Derived Pancreatic Progenitors by Using Human Fetal Pancreatic Mesenchymal Cells in a Micro-scalable Three-Dimensional Co-culture System.

Mesenchymal cells of diverse origins differ in gene and protein expression besides producing varying effects on their organ-matched epithelial cells' maintenance and differentiation capacity. Co-culture with rodent's tissue-specific pancreatic mesenchyme accelerates proliferation, self-renewal, and differentiation of pancreatic epithelial progenitors. Therefore, in our study, the impact of three-dimensional (3D) co-culture of human fetal pancreatic-derived mesenchymal cells (hFP-MCs) with human embryonic stem cell-derived pancreatic progenitors (hESC-PPs) development towards endocrine and beta cells was assessed. Besides, the ability to maintain scalable cultures combining hFP-MCs and hESC-PPs was investigated. hFP-MCs expressed many markers in common with bone marrow-derived mesenchymal stem cells (BM-MSCs). However, they showed higher expression of DESMIN compared to BM-MSCs. After co-culture of hESC-PPs with hFP-MCs, the pancreatic progenitor (PP) spheroids generated in Matrigel had higher expression of NGN3 and INSULIN than BM-MSCs co-culture group, which shows an inductive impact of pancreatic mesenchyme on hESC-PPs beta-cells maturation. Pancreatic aggregates generated by forced aggregation through scalable AggreWell system showed similar features compared to the spheroids. These aggregates, a combination of hFP-MCs and hESC-PPs, can be applied as an appropriate tool for assessing endocrine-niche interactions and developmental processes by mimicking the pancreatic tissue.

Mesenchymal stem cell-derived extracellular vesicles alone or in conjunction with a SDKP-conjugated self-assembling peptide improve a rat model of myocardial infarction.

PURPOSE: The aim of this study was to investigate the cardiac repair effect of human bone marrow mesenchymal stromal cells-derived extracellular vesicles (MSC-EVs) after intramyocardial injection in free form or encapsulated within a self-assembling peptide hydrogel modified with SDKP motif, in a rat model of myocardial infarction (MI). METHODS: MSC-EVs were isolated by ultracentrifuge and characterized for physical parameters and surface proteins. Furthermore, cellular uptake and cardioprotective effects of MSC-EVs were evaluated in vitro using neonatal mouse cardiomyocytes (NMCMs). In vivo effects of MSC-EVs on cardiac repair were studied in rat MI model by comparing the vehicle group (injected with PBS), EV group (injected with MSC-EVs) and Gel + EV group (injected with MSC-EVs encapsulated in (RADA)4-SDKP hydrogel) with respect to cardiac function and fibrotic area using echocardiography and Masson's trichrome staining, respectively. Histological sections were assessed by α-SMA and CD68 immunostaining to investigate the angiogenic and anti-inflammatory effects of the MSC-EVs. RESULTS: We observed the uptake of MSC-EVs into NMCMs which led to NMCMs protection against H2O2-induced oxidative stress by substantial reduction of apoptosis. In myocardial infarcted rats, cardiac function was improved after myocardial injection of MSC-EVs alone or in conjunction with (RADA)4-SDKP hydrogel. This functional restoration coincided with promotion of angiogenesis and decrement of fibrosis and inflammation. CONCLUSION: These data demonstrated that MSC-EVs can be used alone as a potent therapeutic agent for improvement of myocardial infarction.

Human mesenchymal stem cells-conditioned medium improves diabetic wound healing mainly through modulating fibroblast behaviors.

Fibroblast plays a key role in wound healing, and the advantages of mesenchymal stem cells (MSC) secretome in wound healing have previously been reported. In the present study, we investigated the impact of human bone marrow MSC-conditioned media (CM) on skin wound healing in diabetic rats and found that some improvements occurred mainly through fibroblast functions. Then, we scrutinized the impact of MSC-CM treatment on fibroblast cellular behavior by culturing human dermal fibroblasts (HDFs) in a high-glucose (HG) medium, as an in vitro diabetic model. In vivo findings revealed significant improvements in some healing kinetics of diabetic wound which received MSC-CM. Particularly, MSC-CM-treated diabetic wounds reached considerably higher percentages of wound closure. Also, the granulation tissue of these wound had less pronounced inflammatory response, better tissue remodeling, and more vascularization compared with non-treated diabetic ones. Gene expression analyses indicated that MSC-CM treatment leads to upregulation of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) genes. In addition, a significantly higher cell viability/proliferation, migration, and bFGF gene expression were observed when MSC-CM was used to treat HDFs in HG culture media. Based on these findings, it is suggested that MSC-CM could promote wound repair and skin regeneration, in some major processes, via improvement of cellular behaviors of fibroblasts in the diabetic microenvironment. The beneficial advantages of mesenchymal stem cells-conditioned media on fibroblast cellular behaviors and wound healing may lead to establish a novel approach as an alternative therapeutic procedure to cure chronic wounds in diabetic conditions.

Blockage of the Epithelial-to-Mesenchymal Transition Is Required for Embryonic Stem Cell Derivation.

Pluripotent cells emanate from the inner cell mass (ICM) of the blastocyst and when cultivated under optimal conditions immortalize as embryonic stem cells (ESCs). The fundamental mechanism underlying ESC derivation has, however, remained elusive. Recently, we have devised a highly efficient approach for establishing ESCs, through inhibition of the MEK and TGF-ß pathways. This regimen provides a platform for dissecting the molecular mechanism of ESC derivation. Via temporal gene expression analysis, we reveal key genes involved in the ICM to ESC transition. We found that DNA methyltransferases play a pivotal role in efficient ESC generation. We further observed a tight correlation between ESCs and preimplantation epiblast cell-related genes and noticed that fundamental events such as epithelial-to-mesenchymal transition blockage play a key role in launching the ESC self-renewal program. Our study provides a time course transcriptional resource highlighting the dynamics of the gene regulatory network during the ICM to ESC transition.

Allogeneic cell therapy manufacturing: process development technologies and facility design options.

INTRODUCTION: Currently, promising outcomes from clinical trials of allogeneic cells, especially allogeneic mesenchymal stromal cells, fibroblasts, keratinocytes, and human cardiac stem cells, have encouraged research institutions, small and medium enterprises (SMEs), and big pharmaceutical companies to invest and focus on developing allogeneic cell therapy products. Commercial and large-scale production of allogeneic cell therapy products requires unique capabilities to develop technologies that generate safe and effective allogeneic cells/cell lines and their fully characterized master/working banks. In addition, it is necessary to design robust upstream and downstream manufacturing processes, and establish integrated, well-designed manufacturing facilities to produce high quality affordable products in accordance with current GMP regulations for the production of cell therapy products. Areas covered: The authors highlight: the recent advances in the development of allogeneic products, the available options to develop robust manufacturing processes, and facility design considerations. Expert opinion: Currently, there are multiple challenges in development of allogeneic cell therapy products. Indeed, the field is still in its infancy; with technologies and regulations still under development, as is our understanding of the mechanisms of action in the body and their interaction with the host immune system. Their characterization and testing is also an emerging and very complex area.

Novel spliced variants of OCT4, OCT4C and OCT4C1, with distinct expression patterns and functions in pluripotent and tumor cell lines.

OCT4 is a major regulator of pluripotency which has several spliced variants and expressed pseudogenes. Here, we are reporting the existence of two additional novel spliced variants of OCT4, OCT4C and OCT4C1, which lack Exon1 (E1) but start at a novel exon (E0) located ∼14kb upstream of E2. OCT4C/C1 is highly expressed in ES and iPS cells, and their expression was sharply turned off, upon the induction of neural differentiation. The long non-coding RNA (lncRNA) PSORS1C3, is located ∼9kb downstream of the E0 of OCT4C/C1. PSORS1C3 is vigorously spliced to generate nine novel variants, however, none of its exons incorporated in alternatively spliced variants of OCT4. Interestingly, the exons of OCT4 and PSORS1C3 are intertwined, with a novel exon (E0) of PSORS1C3 located ∼4kb upstream of OCT4 E0. This exon participates in generating some more variants of PSORS1C3 (variants 10-24). OCT4C/C1 knock-down in ES and iPS cell lines caused a slight downregulation of PSORS1C3 and OCT4A, a slight upregulation of OCT4B1, and a dramatic upregulation of OCT4B. Altogether, our data revisited the current view of OCT4 gene structure and regulation, and revealed its complex genomic features and expression regulation in stem and tumor cells.

Suppression of transforming growth factor ß signaling promotes ground state pluripotency from single blastomeres.

STUDY QUESTION: Can transforming growth factor ß (TGFß) inhibition promote ground state pluripotency of embryonic stem cells (ESCs) from single blastomeres (SBs) of cleavage embryos in different mouse stains? SUMMARY ANSWER: Small molecule suppression of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and TGFß signaling (designated as R2i) can enhance the generation of mouse ESCs from SBs of different cleavage stage embryos compared with the dual suppression of ERK1/2 and glycogen synthase kinase 3 (GSK3), designated as 2i, regardless of the strain of mouce. WHAT IS KNOWN ALREADY: It is known that chemical inhibition of TGFß promotes ground state pluripotency in the generation and sustenance of naïve ES cells from mouse blastocysts compared with the well-known 2i condition. However, the positive effect of this inhibition on mouse ESCs from early embryonic SBs remains obscure. STUDY DESIGN, SIZE, DURATION: We used 155 cleavage-stage mouse embryos to optimize the culture conditions for blastocyst development. Then, to assess the effects of R2i and 2i on ESC generation from SBs, we cultured isolated SBs in 2i and R2i for 10 days. SBs were replated under the same conditions to produce ESCs. In total, 46 embryos and 321 SBs from two- to eight-cell stages were recovered from NMRI and BALB/c mouse strains and used in this study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blastomeres from 2- to 8-cell stage mouse embryos were dispersed and individually seeded into a 96-well plates that included mitotically inactivated feeder cells. ESCs were generated in B27N2 defined medium supplemented with R2i or 2i. Randomly selected ESC lines, generated from SBs of each stage, were assessed for pluripotency and germ-line transmission. MAIN RESULTS AND THE ROLE OF CHANCE: We demonstrated that dual inhibition of ERK1/2 and TGFß (R2i) enhanced efficient blastocyst development and efficient establishment of ESCs from SB of 2- to 8-cell stage mouse embryos compared with the dual inhibition of ERK1/2 and GSK3 (2i) regardless of the embryonic stage and strain of mice. The proportions of SBs that produced ESC were 50-60 versus 20-30%. LIMITATIONS, REASONS FOR CAUTION: This study was done with mouse embryos, it is not known whether these findings are transferable to humans. WIDER IMPLICATIONS OF THE FINDINGS: These findings resulted in an increased efficiency of ESC generation from one biopsied blastomere for autogeneic or allogeneic matched pluripotent cells without the need to destroy viable embryos. The results also provided information about the developmental capacity of early embryonic blastomeres. STUDY FUNDING/COMPETING INTERESTS: This study was funded by grants provided from Royan Institute, the Iranian Council of Stem Cell Research and Technology and the Iran National Science Foundation. The authors have no conflict of interest to declare.

Models for elastic shells with incompatible strains.

The three-dimensional shapes of thin lamina, such as leaves, flowers, feathers, wings, etc., are driven by the differential strain induced by the relative growth. The growth takes place through variations in the Riemannian metric given on the thin sheet as a function of location in the central plane and also across its thickness. The shape is then a consequence of elastic energy minimization on the frustrated geometrical object. Here, we provide a rigorous derivation of the asymptotic theories for shapes of residually strained thin lamina with non-trivial curvatures, i.e. growing elastic shells in both the weakly and strongly curved regimes, generalizing earlier results for the growth of nominally flat plates. The different theories are distinguished by the scaling of the mid-surface curvature relative to the inverse thickness and growth strain, and also allow us to generalize the classical Föppl-von Kármán energy to theories of prestrained shallow shells.

Differentiation of human embryonic stem cells to hepatocyte-like cells on a new developed xeno-free extracellular matrix.

Human embryonic stem cells (hESCs) provide a new source for hepatocyte production in translational medicine and cell replacement therapy. The reported hESC-derived hepatocyte-like cells (HLCs) were commonly generated on Matrigel, a mouse cell line-derived extracellular matrix (ECM). Here, we performed the hepatic lineage differentiation of hESCs following a stepwise application of growth factors on a newly developed serum- and xeno-free, simple and cost-benefit ECM, designated "RoGel," which generated from a modified conditioned medium of human fibroblasts. In comparison with Matrigel, the differentiated HLCs on both ECMs expressed similar levels of hepatocyte-specific genes, secreted α-fetoprotein, and metabolized ammonia, showed glycogen storage activity as well as low-density lipoprotein and indocyanine green uptake. The transplantation of hESC-HLCs into the carbon tetrachloride-injured liver demonstrated incorporation of the cells into the host mouse liver and the expression of albumin. The results suggest that the xeno-free and cost-benefit matrix may be applicable in bioartificial livers and also may facilitating a clinical application of human pluripotent stem cell-derived hepatocytes in the future.

The effect of Rho-associated kinase inhibition on the proteome pattern of dissociated human embryonic stem cells.

Rho-associated kinase (ROCK) is an immediate downstream target of the Rho GTPase signaling pathway which participates in transducing the Rho GTPase signal to the actin cytoskeleton, leading to the assembly of focal adhesions and stress fibers. Competitive inhibition of ROCK enhances post-thaw viability, improves cloning efficiency and decreases anoikis in human embryonic stem cells (hESCs). The molecular mechanisms by which ROCK inhibition mediates such responses are largely unknown. We have investigated the effect of Y-27632, a competitive ROCK inhibitor (ROCKi), on the proteome of hESCs. HESCs were exposed to ROCK inhibition directly by the addition of Y-27632 to the culture medium or to the Matrigel substratum. ROCK inhibition significantly increased cell survival and plating efficiency without any changes to the morphology, karyotype, or expression of pluripotency markers. We used a two-dimensional gel electrophoresis (2-DE) coupled with tandem mass spectrometry based protein identification and identified 29 ROCKi responsive proteins. As expected, cytoskeleton-related proteins comprised the major ROCKi responsive proteins. Differential proteomic analysis showed that ROCKi induced upregulation of some actin binding proteins such as tropomyosin, F-actin capping protein (CapZ) and transgelin and downregulation of tubulin. In addition, ROCK inhibition was accompanied by changes in expressions of some chromatin modifying proteins such as SMARCB1, ILF3, and Dpy-30-protein, further suggesting a link between ROCK inhibition and the epigenetic mechanism of gene regulation.

Inhibition of TGFß signaling promotes ground state pluripotency.

Embryonic stem (ES) cells are considered to exist in a ground state if shielded from differentiation triggers. Here we show that FGF4 and TGFß signaling pathway inhibitors, designated R2i, not only provide the ground state pluripotency in production and maintenance of naïve ES cells from blastocysts of different mouse strains, but also maintain ES cells with higher genomic integrity following long-term cultivation compared with the chemical inhibition of the FGF4 and GSK3 pathways, known as 2i. Global transcriptome analysis of the ES cells highlights augmented BMP4 signaling pathway. The crucial role of the BMP4 pathway in maintaining the R2i ground state pluripotency is demonstrated by BMP4 receptor suppression, resulting in differentiation and cell death. In conclusion, by inhibiting TGFß and FGF signaling pathways, we introduce a novel defined approach to efficiently establish the ground state pluripotency.