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Ankylosing spondylitis (AS) is an autoimmune disease with unknown aetiology. To unravel the mechanisms mediating AS pathogenesis, we profiled peripheral blood mononuclear cells (PBMCs) from AS patients and healthy subjects using 10X single-cell RNA sequencing. The frequencies of immune cell subsets were evaluated by flow cytometry. NK cells were purified from PBMCs using isolation kit and were examined for gene expression by RT-qPCR. Plasma levels of cytolytic molecules were examined by enzyme-linked immunosorbent assay. Compared to healthy controls, AS patients showed a significant decrease in total NK cells as well as CD56

Background Notch signaling mediates the committed induced differentiation of ear sensory cells and promotes the formation of a precise arrangement of mosaics between hair cells and supporting cells. Embryonic stem cells (ESCs) are pluripotent stem cells which have the potential to differentiate into cell lines through three germ layers. Therefore, it is necessary to study the effects of regulating Notch receptors and ligand expression on the in vitro differentiation equilibrium of hair cells and supporting cells from ESCs.Methods and Results The temporal ex-pression pattern of Notch ligands and receptors during in vitro hair cell-like cell differentia-tion from human embryonic stem cells (hESCs) was detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Subsequently, pAJ-U6-shRNA-CMV-Puro/GFP recombinant lentiviral vectors, encoding short hairpin RNAs, were used to silence JAG-1, JAG-2, and DLL-1, according to the temporal expression pattern of Notch ligands. Then the effect of each ligand on the in vitro differentiation of hair cells was examined by RT-PCR, immunofluorescence, and scanning electron microscopy (SEM).Conclusions Results showed that JAG-1 played an important role in regulating hESC differentiation to otic progenitors. The individual deletion of JAG-2 or DLL-1 had no significant effect on the differentiation of hair cell-like cells. Although the simultaneous inhibition of both DLL-1 and JAG-2 could increase the number of hair cell-like cells, it decreased the number of supporting cells.

TRPM7 is a member of the transient receptor potential cation channel (TRP channel) subfamily M and possesses both an ion channel domain and a functional serine/threonine α-kinase domain. It has been proven to play an essential role in the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs). However, the signaling pathway and molecular mechanism for TRPM7 in regulating osteogenic differentiation remain largely unknown. In this study, the potential role and mechanism of TRPM7 in the osteogenic differentiation of hBMSCs were investigated. The results showed that the expression of TRPM7 mRNA and protein increased, as did the osteogenic induction time. Upregulation or inhibition of TRPM7 could promote or inhibit the osteogenic differentiation of hBMSCs for 14 days. It was also found that the upregulation or inhibition of TRPM7 promoted or inhibited the activity of PLC and SMAD1, respectively, during osteogenic differentiation. PLC could promote osteogenic differentiation by upregulating the activity of SMAD1. However, inhibition of PLC alone could reduce the activity of SMAD1 but not inhibit completely the activation of SMAD1. Therefore, we inferred that it is an important signaling pathway for TRPM7 to upregulate the activity of SMAD1 through PLC and thereby promote the osteogenic differentiation of hBMSCs, but it is not a singular pathway. TRPM7 may also regulate the activation of SMAD1 through other ways, except for PLC, during osteogenic differentiation of hBMSCs.

Microgravity impacts various aspects of human health. Yet the mechanisms of spaceflight-induced health problems are not elucidated. Here, we mapped the fusion systemic analysis of the serum metabolome and the circulating microRNAome in a hindlimb unloading rat model to simulate microgravity. The response of serum metabolites and microRNAs to simulated microgravity was striking. Integrated pathway analysis of altered serum metabolites and target genes of the significantly altered circulating miRNAs with Integrated Molecular Pathway-Level Analysis (IMPaLA) software was mainly suggestive of modulation of neurofunctional signaling pathways. Particularly, we revealed significantly increased miR-383-5p and decreased aquaporin 4 (AQP4) in the hippocampus. Using rabies virus glycoprotein-modified exosomes, delivery of miR-383-5p inhibited the expression of AQP4 not only in rat C6 glioma cells in vitro but also in the hippocampus in vivo. Using bioinformatics to map the crosstalk between the circulating metabolome and miRNAome could offer opportunities to understand complex biological systems under microgravity. Our present results suggested that the change of miR-383-5p level and its regulation of target gene AQP4 was one of the potential molecular mechanisms of microgravity-induced cognitive impairment in the hippocampus.

CHARGE syndrome is a rare disease caused by a genetic disorder. The clinical features of this syndrome include coloboma of the eye, heart anomaly, choanal atresia, retardation of mental and somatic development, microphallus, ear abnormalities and/or deafness. CHD7 is the main causative gene for CHARGE syndrome. In this study, we generated an induced pluripotent stem cell (iPSC) line from the dermal fibroblasts of a 1.5-year-old girl, carrying a de novo mutation (CHD7;NM_017780;c.3449_3450delTC;p.L1151Gfs*17). This iPSC line will be a useful tool for investigating the pathogenesis and for developing treatment for this complicated syndrome.

Heredity is the major factor contributing to the susceptibility to ankylosing spondylitis(AS). Janus kinase 2 (JAK2) has been associated with AS. Urine-derived cells from an AS patient with JAK2 mutation were used to generate induced pluripotent stem cells (iPSCs) with five episomal iPSC reprogramming vectors (pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, pCXLE-hUL, pCXLE-EGFP and pCXWB-EBNA1). The iPSCs were pluripotent and will be valuable for research on the role and mechanism of JAK2 in the pathogenesis of AS.

Background Inner ear hair cells as mechanoreceptors are extremely important for hearing. Defects in hair cells are a major cause of deafness. Induced pluripotent stem cells (iPSCs) are promising for regenerating inner ear hair cells and treating hearing loss. Here, we investigated migration, differentiation, and synaptic connections of transplanted otic epithelial progenitors (OEPs) derived from human iPSCs in mouse cochlea. Methods Human urinary cells isolated from a healthy donor were reprogramed to form iPSCs that were induced to differentiate into OEPs and hair cell-like cells. Immunocytochemistry, electrophysiological examination, and scanning electron microscopy were used to examine characteristics of induced hair cell-like cells. OEP-derived hair cell-like cells were cocultured with spiral ganglion neurons (SGNs), and the markers of synaptic connections were detected using immunocytochemistry and transmission electron microscope. In vivo, OEPs derived from iPSCs were transplanted into the cochlea of mice by injection through the round window. Migration, differentiation, and synaptic connections of transplanted cells were also examined by thin cochlear sectioning and immunohistochemistry. Results The induced hair cell-like cells displayed typical morphological characteristics and electrophysiological properties specific to inner hair cells. In vitro, OEP-derived hair cell-like cells formed synaptic connections with SGNs in coculture. In vivo, some of the transplanted cells migrated to the site of the resident hair cells in the organ of Corti, differentiated into hair cell-like cells, and formed synaptic connections with native SGNs. Conclusions We conclude that the transplantation of OEPs is feasible for the regeneration of hair cells. These results present a substantial reference for a cell-based therapy for the loss of hair cells.

Bone formation is linked with osteogenic differentiation of mesenchymal stem cells (MSCs) in the bone marrow. Microgravity in spaceflight is known to reduce bone formation. In this study, we used a real microgravity environment of the SJ-10 Recoverable Scientific Satellite to examine the effects of space microgravity on the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs). hMSCs were induced toward osteogenic differentiation for 2 and 7 d in a cell culture device mounted on the SJ-10 satellite. The satellite returned to Earth after going through space experiments in orbit for 12 d, and cell samples were harvested and analyzed for differentiation potentials. The results showed that space microgravity inhibited osteogenic differentiation and resulted in adipogenic differentiation, even under osteogenic induction conditions. Under space microgravity, the expression of 10 genes specific for osteogenesis decreased, including collagen family members, alkaline phosphatase ( ALP), and runt-related transcription factor 2 ( RUNX2), whereas the expression of 4 genes specific for adipogenesis increased, including adipsin ( CFD), leptin ( LEP), CCAAT/enhancer binding protein β ( CEBPB), and peroxisome proliferator-activated receptor-γ ( PPARG). In the analysis of signaling pathways specific for osteogenesis, we found that the expression and activity of RUNX2 was inhibited, expression of bone morphogenetic protein-2 ( BMP2) and activity of SMAD1/5/9 were decreased, and activity of focal adhesion kinase (FAK) and ERK-1/2 declined significantly under space microgravity. These data indicate that space microgravity plays a dual role by decreasing RUNX2 expression and activity through the BMP2/SMAD and integrin/FAK/ERK pathways. In addition, we found that space microgravity increased p38 MAPK and protein kinase B (AKT) activities, which are important for the promotion of adipogenic differentiation of hMSCs. Space microgravity significantly decreased the expression of Tribbles homolog 3 ( TRIB3), a repressor of adipogenic differentiation. Y15, a specific inhibitor of FAK activity, was used to inhibit the activity of FAK under normal gravity; Y15 decreased protein expression of TRIB3. Therefore, it appears that space microgravity decreased FAK activity and thereby reduced TRIB3 expression and derepressed AKT activity. Under space microgravity, the increase in p38 MAPK activity and the derepression of AKT activity seem to synchronously lead to the activation of the signaling pathway specifically promoting adipogenesis.-Zhang, C., Li, L., Jiang, Y., Wang, C., Geng, B., Wang, Y., Chen, J., Liu, F., Qiu, P., Zhai, G., Chen, P., Quan, R., Wang, J. Space microgravity drives transdifferentiation of human bone marrow-derived mesenchymal stem cells from osteogenesis to adipogenesis.

Inner ear hair cells are mechanoreceptors responsible for hearing. Pathogenic defects of hair cell-specific genes are one of the major causes of deafness. The BarH class homeobox gene Barhl1 is a deafness gene expressed in developing hair cells, yet the role of Barhl1 during hair cell development remains poorly understood. In the present study, we first established an in vitro differentiation system to efficiently obtain mouse embryonic stem cell (mESC)-derived hair cell-like cells. Subsequently, a mESC line carrying a targeted disruption of Barhl1 was generated using CRISPR/Cas9 technology and subjected to the established in vitro hair cell differentiation protocol. Targeted disruption of Barhl1 does not affect the induction of mESCs toward early primitive ectoderm-like (EPL) cells and otic progenitors but strongly inhibits the differentiation of hair cell-like cells. Using RNA-sequencing and bioinformatics, we further unravel the molecular mechanism underlying Barhl1-mediated hair cell development. Our data demonstrate the essential role of Barhl1 during hair cell development and provide a basis for the treatment of Barhl1 mutation-based deafness.

Sensorineural hearing loss and vestibular dysfunction have become the most common forms of sensory defects. Stem cell-based therapeutic strategies for curing hearing loss are being developed. Several attempts to develop hair cells by using chicken utricle stromal cells as feeder cells have resulted in phenotypic conversion of stem cells into inner ear hair-cell-like cells. Here, we induced the differentiation of human embryonic stem cells (hESCs) into otic epithelial progenitors (OEPs), and further induced the differentiation of OEPs into hair-cell-like cells using different substrates. Our results showed that OEPs cultured on the chicken utricle stromal cells with the induction medium could differentiate into hair-cell-like cells with stereociliary bundles. Co-culture with stromal cells, however, may be problematic for subsequent examination of the induced hair-cell-like cells. In order to avoid the interference from stromal cells, we cultured OEPs on laminin with different induction media and examined the effects of the induction medium on the differentiation potentials of OEPs into hair-cell-like cells. The results revealed that the culture of OEPs on laminin with the conditioned medium from chicken utricle stromal cells supplemented with EGF and all-trans retinoic acid (RA) could promote the organization of cells into epithelial clusters displaying hair-cell-like cells with stereociliary bundles. These cells also displayed the expected electrophysiological properties.

Background Induced pluripotent stem cells (iPSCs) can generate epithelial stem cells (EpSCs) as seed cells for skin substitutes to repair skin defects. Here, we investigated the effects of a human acellular amniotic membrane (hAAM) combined with iPSC-derived CD200+/ITGA6+ EpSCs as a skin substitute on repairing skin defects in nude mice. Methods Human urinary cells isolated from a healthy donor were reprogrammed into iPSCs and then induced into CD200+/ITGA6+ epithelial stem cells. Immunocytochemistry and RT-PCR were used to examine the characteristics of the induced epithelial stem cells. iPSC-derived EpSCs were cultured on a hAAM, and cytocompatibility of the composite was analyzed by CCK8 assays and scanning electron microscopy. Then, hAAMs combined with iPSC-derived EpSCs were transplanted onto skin defects of mice. The effects of this composite on skin repair were evaluated by immunohistochemistry. Results The results showed that CD200+/ITGA6+ epithelial stem cells induced from iPSCs displayed the phenotypes of hair follicle stem cells. After seeding on the hAAM, iPSC-derived epithelial stem cells had the ability to proliferate. After transplantation, CD200+/ITGA6+ epithelial stem cells on the hAAM promoted the construction of hair follicles and interfollicular epidermis. Conclusions These results indicated that transplantation of a hAAM combined with iPS-derived EpSCs is feasible to reconstruct skin and skin appendages, and may be a substantial reference for iPSC-based therapy for skin defects.

With the development of scientific exploration in deep space, human activities will become more frequent, and activity time will be longer in the deep space. The environment alteration may cause severe bone changes of human in deep space. The changes of bone mass caused by spatial microgravity are related to the decrease of osteoblast formation and development in bone tissue, and the decrease of osteoblast formation is related to the down-regulation of differentiation of human bone marrow mesenchymal stem cells (hMSCs). Therefore, the study for the biological effects of microgravity on bone cell formation and the relative molecular mechanisms at stem cell level is one of the important subjects to explore the effects of spatial microgravity on bone changes. These studies may provide a scientific basis for the development and the related technologies of target drugs research. Based on exploring the flight conditions on the ground and simulating flight experiments with the automated space experimental device, we utilized a real microgravity environment in the SJ-10 recoverable microgravity experimental satellite (SJ-10 satellite) to examine the effects of space microgravity on transcriptome expression and differentiation potentials of hMSCs.

Damage or loss of auditory hair cells leads to irreversible sensorineural hearing loss in human, thus regeneration of these cells to reconstruct auditory sensory epithelium holds the promise for the treatment of deafness. Regulatory factors involved in the development of auditory sensory epithelium play crucial roles in hair cell regeneration and hearing restoration. Here, we first focus on the transcription factor Atoh1 which is critical for hair cell development and regeneration, and comprehensively summarize the current understanding of the protein structure, target binding motif, developmental expression pattern, functional role, and upstream and downstream regulatory mechanism of Atoh1 in the context of controlling the cell fate commitment to hair cells or transdifferentiation from supporting cells. We also discuss cellular context dependency of Atoh1 in hair cell induction which should be taken into consideration when using Atoh1 gene therapy for hair cell regeneration. Next, we review the roles of Gfi1, Pou4f3, and Barhl1 in hair cell maturation and maintenance, and suggest that manipulation of these genes and their downstream targets will be helpful for the generation of functional hair cells with long-term viability. Finally, we provide an overview of the interplay between Notch, Wnt, Shh, and FGF signaling pathways during auditory sensory epithelium development. By analyzing crosstalk between these pathways, we suggest that combination of Wnt signaling activation with Hey1 and Hey2 inhibition will be crucial for hair cell regeneration and hearing restoration. Furthermore, this review highlights the importance of deeper understanding of the cellular context for hair cell development and the interconnection between these key regulators in developing new strategies to treat sensorineural hearing loss.

Mesenchymal stem cells (MSCs) may have potential applications in regenerative medicine for the treatment of chronic liver diseases (CLDs). Human menstrual blood is a novel source of MSCs, termed menstrual blood-derived stem cells (MenSCs). Compared with bone marrow MSCs, MenSCs exhibit a higher proliferation rate and they can be obtained through a simple, safe, painless procedure without ethical concerns. Although the therapeutic efficacy of MenSCs has been explored in some diseases, their effects on liver fibrosis are still unclear. In the present study, we investigated the therapeutic effects of MenSC transplantation in a carbon tetrachloride-induced mouse model of liver fibrosis. These results revealed that MenSCs markedly improved liver function, attenuated collagen deposition, and inhibited activated hepatic stellate cells up to 2 weeks after transplantation. Moreover, tracking of green fluorescent protein-expressing MenSCs demonstrated that transplanted cells migrated to the sites of injury, but few differentiated into functional hepatocyte-like cells. Transwell coculturing experiments also showed that MenSCs suppressed proliferation of LX-2 cells (an immortalized hepatic stellate cell line) through secretion of monocyte chemoattractant protein-1, interleukin-6, hepatocyte growth factor, growth-related oncogene, interleukin-8, and osteoprotegerin. Collectively, our results provided preliminary evidence for the antifibrotic capacity of MenSCs in liver fibrosis and suggested that these cells may be an alternative therapeutic approach for the treatment of CLDs.

Deafness or hearing loss is a major issue in human health. Inner ear hair cells are the main sensory receptors responsible for hearing. Defects in hair cells are one of the major causes of deafness. A combination of induced pluripotent stem cell (iPSC) technology with genome-editing technology may provide an attractive cell-based strategy to regenerate hair cells and treat hereditary deafness in humans. Here, we report the generation of iPSCs from members of a Chinese family carrying MYO15A c.4642G>A and c.8374G>A mutations and the induction of hair cell-like cells from those iPSCs. The compound heterozygous MYO15A mutations resulted in abnormal morphology and dysfunction of the derived hair cell-like cells. We used a CRISPR/Cas9 approach to genetically correct the MYO15A mutation in the iPSCs and rescued the morphology and function of the derived hair cell-like cells. Our data demonstrate the feasibility of generating inner ear hair cells from human iPSCs and the functional rescue of gene mutation-based deafness by using genetic correction.

Objectives Exposure to microgravity induces many adaptive and pathological changes in human bone marrow mesenchymal stem cells (hBMSCs). However, the underlying mechanisms of these changes are poorly understood. We revealed the gene expression patterns of hBMSCs under normal ground (NG) and simulated microgravity (SMG), which showed an interpretation for these changes by gene regulation and signal pathways analysis. Materials and methods In this study, hBMSCs were osteogenically induced for 0, 2, 7 and 14 days under normal ground gravity and simulated microgravity, followed by analysis of the differences in transcriptome expression during osteogenic differentiation by RNA sequencing and some experimental verification for these results. Results The results indicated that 837, 399 and 894 differentially expressed genes (DEGs) were identified in 2, 7 and 14 days samples, respectively, out of which 13 genes were selected for qRT‐PCR analysis to confirm the RNA‐sequencing results. After analysis, we found that proliferation was inhibited in the early stage of induction. In the middle stage, osteogenic differentiation was inhibited, whereas adipogenic differentiation benefited from SMG. Moreover, SMG resulted in the up‐regulation of genes specific for tumorigenesis in the later stage. Conclusion Our data revealed that SMG inhibits the proliferation and inhibits the differentiation towards osteoblasts but promotes adipogenesis. SMG also selects highly tumorigenic cells for survival under prolonged SMG.

With rapid development of space engineering, research on life sciences in space is being conducted extensively, especially cellular and molecular studies on space medicine. Stem cells, undifferentiated cells that can differentiate into specialized cells, are considered a key resource for regenerative medicine. Research on stem cells under conditions of microgravity during a space flight or a ground-based simulation has generated several excellent findings. To help readers understand the effects of outer space and ground-based simulation conditions on stem cells, we reviewed recent studies on the effects of microgravity (as an obvious environmental factor in space) on morphology, proliferation, migration, and differentiation of stem cells.

The induction of inner ear hair cells from stem cells or progenitor cells in the inner ear proceeds through a committed inner ear sensory progenitor cell stage prior to hair cell differentiation. To increase the efficacy of inducing inner ear hair cell differentiation from the stem cells or progenitor cells, it is essential to identify comprehensive markers for the stem cells/progenitor cells from the inner ear, the committed inner ear sensory progenitor cells and the differentiating hair cells to optimize induction conditions. Here, we report that we efficiently isolated and expanded the stem cells or progenitor cells from postnatal mouse cochleae, and induced the generation of inner ear progenitor cells and subsequent differentiation of hair cells. We profiled the gene expression of the stem cells or progenitor cells, the inner ear progenitor cells, and hair cells using aRNA microarray analysis. The pathway and gene ontology (GO) analysis of differentially expressed genes was performed. Analysis of genes exclusively detected in one particular cellular population revealed 30, 38, and 31 genes specific for inner ear stem cells, inner ear progenitor cells, and hair cells, respectively. We further examined the expression of these genes in vivo and determined that Gdf10+Ccdc121, Tmprss9+Orm1, and Chrna9+Espnl are marker genes specific for inner ear stem cells, inner ear progenitor cells, and differentiating hair cells, respectively. The identification of these marker genes will likely help the effort to increase the efficacy of hair cell induction from the stem cells or progenitor cells.

Osteogenic differentiation in human bone marrow-derived mesenchymal stem cells (hBMSCs) is regulated by various factors, including bone morphogenetic proteins (BMPs), Notch, growth hormones and mitogen-activated protein kinases (MAPKs). Tribbles homolog 3 (TRIB3), a pseudokinase, plays an important role in cancer cells and adipocytes. However, TRIB3 function in osteogenic differentiation is unknown, although it is involved in regulating signaling pathways associated with osteogenic differentiation. Here, we found that TRIB3 was highly expressed during osteogenic differentiation in hBMSCs. Inhibition of focal adhesion kinase (FAK) or phosphatidylinositol 3-kinase (PI3K) resulted in a significant decrease in TRIB3 expression, and expression of TRIB3 was restored by increasing insulin-like growth factor-1 (IGF-1) via activating phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling. TRIB3 knock-down enhanced proliferation and decreased osteogenic differentiation at the middle stage of differentiation, and these effects were reversed by inhibiting the activation of extracellular signal-regulated kinase (ERK)-1/2. In conclusion, TRIB3 plays an important role in proliferation and osteogenic differentiation by regulating ERK1/2 activity at the middle stage of differentiation, and expression of TRIB3 is regulated by FAK in a PI3K/AKT-dependent manner.

Hindlimb unloading, as a simulation of microgravity, decreases the osteogenic potential of mesenchymal stem cells (MSCs) from hindlimb femur of rat. We simulated the microgravity by 28-day of hindlimb unloading for male Sprague-Dawley rat, and performed intramuscular injection of BMP-2 and FGF2 at a given interval during hindlimb unloading. Then, the bone marrow (BM) was collected from hindlimb femur of rat. MSCs were isolated from BM, cultured for four passages, and then induced for osteogenesis. The results revealed that the hindlimb unloading decreased the osteogenic potential of MSCs and also the expression of osteoblast gene marker mRNAs in cells induced by osteogenic conditions. Hindlimb unloading for 28 days resulted in the decrease of vinculin-containing focal adhesion in MSCs. During hindlimb unloading, the interval intramuscular injection of BMP-2 or FGF2 alone could increase the osteogenic potential of MSCs and the expression of osteoblast gene marker mRNA. However, the effect of BMP-2 or FGF2 injection alone was significantly lower than that of combination injection of both factors. The further examination showed that the intramuscular injection of BMP-2 promoted the expression of Runx2 mRNA and that the intramuscular injection of FGF2 increased the phosphorylation of ERK and Runx2. Nevertheless, the intramuscular injection of any factor could not increase the formation of vinculin-containing focal adhesions in MSCs. This suggests that BMP-2 should increase the expression of Runx2, and that the activation of Runx2 should be promoted by the FGF2 signaling pathway which activated ERK/Runx2. The activation of this signaling pathway should not lie on the formation of vinculin-containing focal adhesions.

In mammals, hair cells may be damaged or lost due to genetic mutation, infectious disease, chemical ototoxicity, noise and other factors, causing permanent sensorineural deafness. Regeneration of hair cells is a basic pre-requisite for recovery of hearing in deaf animals. The inner ear stem cells in the organ of Corti and vestibular utricle are the most ideal precursors for regeneration of inner ear hair cells. This review highlights some recent findings concerning the proliferation and differentiation of inner ear stem cells. The differentiation of inner ear stem cells into hair cells involves a series of signaling pathways and regulatory factors. This paper offers a comprehensive analysis of the related studies.

In this paper, we developed a two-step-induction method of generating functional hair cells from inner ear multipotent cells. Multipotent cells from the inner ear were established and induced initially into progenitor cells committed to the inner ear cell lineage on the poly-L-lysine substratum. Subsequently, the committed progenitor cells were cultured on the mitotically inactivated chicken utricle stromal cells and induced into hair-cell-like cells containing characteristic stereocilia bundles. The hair-cell-like cells exhibited rapid permeation of FM1-43FX. The whole-cell patch-clamp technique was used to measure the membrane currents of cells differentiated for 7 days on chicken utricle stromal cells and analyze the biophysical properties of the hair-cell-like cells by recording membrane properties of cells. The results suggested that the hair-cell-like cells derived from inner ear multipotent cells were functional following differentiation in an enabling environment.

Osteoporosis is a common disease in the elderly population. The progress of this disease results in the reduction of bone mass and can increase the incidence of fractures. Drugs presently used clinically can block the aggravation of this disease. However, these drugs cannot increase the bone mass and may result in certain side effects. Statins, also known as HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase inhibitors, have been widely prescribed for CVD (cardiovascular disease) for decades. Nonetheless, several studies have demonstrated that statins exert bone anabolic effect and may be helpful for the treatment of osteoporosis. Several experiments have analysed the mechanisms of bone anabolism regulated by statins. In the present paper, we review the mechanisms of promoting osteogenesis, suppressing osteoblast apoptosis and inhibiting osteoclastogenesis.

Epac, exchange protein activated by cyclic adenosine monophosphate (cAMP), could regulate the trans-differentiation between adipogenesis and osteogenesis of human mesenchymal stem cells (hMSCs). Epac activated by 8-pCPT-2′- O -Me-cAMP, a cAMP analog preferentially activating Epac, resulted in the increase of adipogenic gene expression and the decrease of osteogenic gene expression. The pro-adipogenic and anti-osteogenic effect of 8-pCPT-2′- O -Me-cAMP was attributed to that 8-pCPT-2′- O -Me-cAMP led to the activation of protein kinase B (PKB) and cAMP response element-binding protein (CREB) as well as the inhibition of Ras homolog gene family member A (RhoA), focal adhesion kinase (FAK), extracellular-signal-regulated kinase (ERK) and runt-related transcription factor 2 (Runx2) activities. Inhibition of Epac by a dominant-negative form of Epac1 resulted in the decrease of phosphatidylinositol 3-kinase (PI3K), PKB and CREB activities as well as down-regulation of peroxisome proliferator activated receptor-γ (PPARγ) expression. Inhibition of PI3K by a specific inhibitor or inhibition of Arf and Rho GAP adapter protein 3 (ARAP3, a phosphatidylinositol (PtdIns)(3,4,5)P 3 binding protein) by ARAP3 siRNA led to the recovery of RhoA and FAK activities. RhoA-V14, a constitutively active form of RhoA, could activate the MEK/ERK/Runx2 signaling. Therefore, we conclude that PI3K activated by Epac leads to the activation of PKB/CREB signaling and the up-regulation of PPARγ expression, which in turn activate the transcription of adipogenic genes; whereas osteogenesis is driven by Rho/FAK/MEK/ERK/Runx2 signaling, which can be inhibited by Epac via PI3K. These results should be helpful to provide new targets for treatment of osteoporosis and related bone-wasting diseases.

Fluid shear stress (FSS) is an important biomechanical factor regulating the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and is therefore widely used in bone tissue engineering. However, the mechanotransduction of FSS in hMSCs remains largely unknown. As β1 integrins are considered to be important mechanoreceptors in other cells, we suspect that β1 integrins should also be important for hMSCs to sense the stimulation of FSS. We used a perfusion culture system to produce FSS loading on hMSCs seeded in PLGA three-dimensional (3D) scaffolds and investigated the roles of β1 integrins, FAK and ERK1/2 in FSS-induced osteogenic differentiation of hMSCs. Our results showed that FSS not only markedly increased ALP activity and the expression of ALP, OCN, Runx2 and COLIα genes but also significantly enhanced the phosphorylation of ERK1/2, Runx2 and FAK. FSS-induced activation of ERK1/2 and FAK was inhibited by blockade of the connection between β1 integrins and ECM with RGDS peptide and integrins β1 monoclonal antibody. Our study also found that FSS could upregulate the expression level of β1 integrins and that this upregulation could be abolished by PD98059. Further investigation indicated that FSS-activated ERK1/2 led to the phosphorylation of IκBα and NFκB p65. The activation of NFκB p65 resulted in the upregulation of β1 integrin expression. Therefore, it could be inferred that β1 integrins should sense the stimulation of FSS and thus activate ERK1/2 through activating of FAK, and FSS-activated ERK1/2 feedback to upregulate the expression of β1 integrins through activating NFκB.

A reasonable mechanical microenvironment similar to the bone microenvironment in vivo is critical to the formation of engineering bone tissues. As fluid shear stress (FSS) produced by perfusion culture system can lead to the osteogenic differentiation of human mesenchymal stem cells (hMSCs), it is widely used in studies of bone tissue engineering. However, effects of FSS on the differentiation of hMSCs largely depend on the FSS application manner. It is interesting how different FSS application manners influence the differentiation of hMSCs. In this study, we examined the effects of intermittent FSS and continuous FSS on the osteogenic differentiation of hMSCs. The phosphorylation level of ERK1/2 and FAK is measured to investigate the effects of different FSS application manners on the activation of signaling molecules. The results showed that intermittent FSS could promote the osteogenic differentiation of hMSCs. The expression level of osteogenic genes and the alkaline phosphatase (ALP) activity in cells under intermittent FSS application were significantly higher than those in cells under continuous FSS application. Moreover, intermittent FSS up-regulated the activity of ERK1/2 and FAK. Our study demonstrated that intermittent FSS is more effective to induce the osteogenic differentiation of hMSCs than continuous FSS.

The goal of this study was to determine the effects of hindlimb unloading (HU) on the ex vivo growth and the osteogenic potential of mesenchymal stem cells (MSCs) from the femurs of rats. Microgravity was simulated by 28-day HU in male Sprague-Dawley (SD) rats, and the bone marrow (BM) was collected from hindlimb femurs of HU or control (CTL) rats. MSCs were isolated from BM and cultured for eight passages. Then MSCs at passages 2, 4, and 8 were induced for osteogenesis or adipogenesis. The results revealed that HU decreased the osteogenic potential of MSCs and also decreased the expression of osteoblast gene marker mRNAs in cells induced by osteogenic conditions. Meanwhile, the expression of Runx2 mRNA and the phosphorylation of ERK were also decreased. There were no significant differences of osteoblast gene marker and Runx2 mRNA expression between cells induced from different passages of MSCs in UH rats. Under adipogenic conditions, HU increased both the adipogenic potential of MSCs and the expression of adipocytic gene marker mRNAs in induced cells. HU also increased the expression of PPAR gamma 2 mRNA, but with no effect on the phosphorylation of p38MAPK. The adipogenic potential of MSCs and the expression of adipocytic gene marker mRNAs in induced cells decreased along with cell cultures under normal gravity. This suggests that the normal gravity during in vitro MSC culture and the centrifugal force produced during cell harvest after each passage could decrease the adipogenic potential of MSCs, but could not reverse the effect of HU on the osteogenic potential of MSCs.

Human mesenchymal stem cells (MSCs) are multipotential and are detected in bone marrow (BM), adipose tissue, placenta, and umbilical cord blood (UCB). In this study, we examined the ability of UCB-derived MSCs (UCB-MSCs) to support ex vivo expansion of hematopoietic stem/progenitor cells (HSPCs) from UCB and the engraftment of expanded HSPCs in NOD/SCID mice. The result showed that UCB-MSCs supported the proliferation and differentiation of CD34+ cells in vitro. The number of expanded total nucleated cells (TNCs) in MSC-based culture was twofold higher than cultures without MSC (control cultures). UCB-MSCs increased the expansion capabilities of CD34+ cells, long-term culture-initiating cells (LTC-ICs), granulocyte-macrophage colony-forming cells (GM-CFCs), and high proliferative potential colony-forming cells (HPP-CFCs) compared to control cultures. The expanded HSPCs were transplanted into lethally irradiated NOD/SCID mice to assess the effects of expanded cells on hematopoietic recovery. The number of white blood cells (WBCs) in the peripheral blood of mice transplanted with expanded cells from both the MSC-based and control cultures returned to pretreatment levels at day 25 posttransplant and then decreased. The WBC levels returned to pretreatment levels again at days 45–55 posttransplant. The level of human CD45+ cell engraftment in primary recipients transplanted with expanded cells from the MSC-based cultures was significantly higher than recipients transplanted with cells from the control cultures. Serial transplantation demonstrated that the expanded cells could establish long-term engraftment of hematopoietic cells. UCB-MSCs similar to those derived from adult bone marrow may provide novel targets for cellular and gene therapy.

This study aimed to investigate the potentials of ex vivo expansion and pluridifferentiation of cryopreserved adult human bone marrow mesenchymal stem cells (hMSCs) into adipocytes and neurocytes. Cryopreserved hMSCs were resuscitated and cultured for 15 passages, and then induced to adipocytes and neurocytes with corresponding induction medium. The induced cells were observed for morphological properties and expression of triglyceride or neuron-specific enolase and nestin was detected. The result showed that the resuscitated cells cultured in induction medium consisting of dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin and insulin-like growth factor I (IGF-I) showed adipogenesis, and lipid vacuole accumulation was detectable after 21days. The resuscitated hMSCs were also induced into neurocytes and expressed nestin and neuron-specific enolase (NSE), which are special surface markers associated with neural cells at different stages. This study suggested that resuscitated hMSCs should still be a population of pluripotential cells and should be accessible for establishing an abundant hMSC reservoir for further experiment and treatment of various clinical diseases.

The genetic correction of induced pluripotent stem cells (iPSCs) induced from somatic cells of patients with sensorineural hearing loss (caused by hereditary factors) is a promising method for its treatment. The correction of gene mutations in iPSCs could restore the normal function of cells and provide a rich source of cells for transplantation. In the present study, iPSCs were generated from a deaf patient with compound heterozygous MYO7A mutations (c.1184G>A and c.4118C>T; P-iPSCs), the asymptomatic father of the patient (MYO7A c.1184G>A mutation; CF-iPSCs), and a normal donor (MYO7AWT/WT; C-iPSCs). One of MYO7A mutation sites (c.4118C>T) in the P-iPSCs was corrected using CRISPR/Cas9. The corrected iPSCs (CP-iPSCs) retained cell pluripotency and normal karyotypes. Hair cell-like cells induced from CP-iPSCs showed restored organization of stereocilia-like protrusions; moreover, the electrophysiological function of these cells was similar to that of cells induced from C-iPSCs and CF-iPSCs. These results might facilitate the development of iPSC-based gene therapy for genetic disorders. Significance Induced pluripotent stem cells (iPSCs) were generated from a deaf patient with compound heterozygous MYO7A mutations (c.1184G>A and c.4118C>T). One of the MYO7A mutation sites (c.4118C>T) in the iPSCs was corrected using CRISPR/Cas9. The genetic correction of MYO7A mutation resulted in morphologic and functional recovery of hair cell-like cells derived from iPSCs. These findings confirm the hypothesis that MYO7A plays an important role in the assembly of stereocilia into stereociliary bundles. Thus, the present study might provide further insight into the pathogenesis of sensorineural hearing loss and facilitate the development of therapeutic strategies against monogenic disease through the genetic repair of patient-specific iPSCs.

A new marrow-derived mesenchymal stem cell (hMSC) line that could support expansion of hematopoietic stem/progenitor cells (HSPCs) was developed. Primary hMSCs were infected with retrovirus containing Flt-3 ligand and thrombopoietin genes. CD34+ cells from cord blood were expanded with primary hMSCs or transduced hMSCs. The expansion of total nucleated cells, CD34+ cells and mixed colonies containing erythroid and myeloid cells and megakaryocytes for 2 weeks coculture with transduced hMSCs was remarkably increased. The outputs of long-term culture-initiating cells for 2 and 4 weeks coculture with transduced hMSCs were also largely increased. The expansion rates of HSPCs with transduced hMSCs were unchanged for 6 weeks. In contrast, the expansion rates of HSPCs with primary hMSCs declined drastically through 6 weeks. SCID-repopulating cell expansion with transduced hMSCs for 4 weeks was significantly higher than that of uncultured CD34,+ cells and HSPCs expanded with primary hMSCs.

Human mesenchymal stem cells (hMSCs) are one of the important factors that regulate bone anabolism. Osteoporosis resulting from microgravity during spaceflight may possibly be due to a decrease in osteogenesis mediated by hMSCs. This speculation should be verified through culture and osteogenic induction of hMSCs in a microgravity environment during spaceflight. Control of CO2 is a key component in current experimental protocols for growth, survival, and proliferation of in vitro cultured cells. However, carrying CO2 tanks on a spaceflight and devoting space/mass allowances for classical CO2 control protocols make experimentation on culture and osteogenesis difficult during most missions. Therefore, an experimental culture and osteogenic medium was developed through modifying the components of buffer salts in conventional culture medium. This experimental medium was used to culture and induce hMSCs under CO2-independent conditions. The results showed that culture and induction of hMSCs with conventional culture medium and conventional osteogenic medium under CO2-independent conditions resulted in an increase of pH in medium. The proliferation of hMSCs was also inhibited. hMSCs cultured with experimental culture medium under CO2-independent conditions showed a proliferation potential that was the same as those cultured with conventional culture medium under CO2-dependent conditions. The experimental osteogenic medium could promote hMSCs to differentiate into osteoblast-like cells under CO2-independent conditions. Cells induced by this induction system showed high alkaline phosphatase activity. The expression levels of osteogenic genes in cells induced with experimental osteogenic medium under CO2-independent conditions were not significantly different from those cells induced with conventional osteogenic medium under CO2-dependent conditions. These results suggest that the experimental culture and induction system could be used to culture hMSCs and induce the osteogenesis of hMSCs in the atmospheric conditions common to spaceflights without additional CO2. Key Words: hMSCs—CO2-independent culture—Osteogenic differentiation—Proliferation. Astrobiology 13, 370–379.

Human mesenchymal stem cells are primary multipotent cells capable of differentiating into several cell types including adipocytes when cultured under defined in vitro conditions. In the present study we investigated the role of cAMP signaling and its downstream effectors, protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac) in adipocyte conversion of human mesenchymal stem cells derived from adipose tissue (hMADS). We show that cAMP signaling involving the simultaneous activation of both PKA- and Epac-dependent signaling is critical for this process even in the presence of the strong adipogenic inducers insulin, dexamethasone, and rosiglitazone, thereby clearly distinguishing the hMADS cells from murine preadipocytes cell lines, where rosiglitazone together with dexamethasone and insulin strongly promotes adipocyte differentiation. We further show that prostaglandin I(2) (PGI(2)) may fully substitute for the cAMP-elevating agent isobutylmethylxanthine (IBMX). Moreover, selective activation of Epac-dependent signaling promoted adipocyte differentiation when the Rho-associated kinase (ROCK) was inhibited. Unlike the case for murine preadipocytes cell lines, long-chain fatty acids, like arachidonic acid, did not promote adipocyte differentiation of hMADS cells in the absence of a PPARγ agonist. However, prolonged treatment with the synthetic PPARδ agonist L165041 promoted adipocyte differentiation of hMADS cells in the presence of IBMX. Taken together our results emphasize the need for cAMP signaling in concert with treatment with a PPARγ or PPARδ agonist to secure efficient adipocyte differentiation of human hMADS mesenchymal stem cells.

It is a classical signaling pathway that the activation of extracellular signal-regulated kinase1/2 (ERK1/2) results in the phosphorylation of runt-related transcription factor 2 (Runx2) and thereby initiates the transcription of osteogenic genes. Recently, it is found that the activation of ERK1/2 resulted from fluid shear stress (FSS) also increased the expression of Runx2 and β1 integrins, and finally enhanced osteogenic differentiation. However, it has been remained largely unknown how ERK1/2 regulates the expression of Runx2 and β1 integrins. We use the perfusion culture system to produce FSS exerting on human bone marrow-derived mesenchymal stem cells (hMSCs) and thus activate ERK1/2. Our study demonstrated that FSS-activated ERK1/2 mediated the expression of osteogenic genes via two novel signaling pathways except for the classical signaling pathway: feedback up-regulation of β1 integrins expression via activating nuclear factor kappa B (NF-κB), and activation of bone morphogenesis proteins (BMPs)/mothers against decapentaplegic (Smad) pathway via activating NF-κB and thereby regulating Runx2 expression. These signaling pathways combined with the classical signaling pathway, with ERK1/2 as a hub node molecule, form a molecular signaling cross-talking network to induce the osteogenic differentiation of hMSCs. The understanding on the mechanism of FSS inducing the osteogenic differentiation of hMSCs will not only be helpful to develop the bone tissue engineering but also provide new targets for drug discovery for treatment of osteoporosis and other related bone-wasting diseases.

The aim of the present study was to investigate the effects of abnormal gravity on human mesenchymal stem cells (hMSCs). Strong magnetic field and magnetic field gradient generate a magnetic force that can add to or subtract from the gravitational force. In this study, this is defined as a high-magneto-gravitational environment (HMGE). The HMGE provides three apparent gravity levels, i.e. hypogravity (μg), hypergravity (2g) and normal gravity with strong magnetic field (1g) conditions. After hMSCs were subject to HMGE for 12 h, the proliferation, morphology, structure and apoptosis were investigated. Results showed that the proliferation of hMSCs was inhibited under μg condition. The abnormal gravity induced morphologic characteristics of apoptosis cells, such as cell shrinkage, membrane blebbing, nuclear chromatin condensation and margination, decreased cell viability, and increased caspase-3/7 activity. The rate of apoptosis under μg condition is up to 56.95%. The F-actin stress fibers and microtubules were disrupted under abnormal gravity condition. Under μg-condition, the expression of p53 at mRNA and protein levels was up-regulated more than 9- and 6 folds, respectively. The Pifithrin-α, an specific inhibitor of p53, inhibited the apoptosis and prevented the disruption of cytoskeleton induced by abnormal gravity. These results implied that hMSCs were sensitive to abnormal gravity and exhibited classic apoptotic features, which might be associated with p53 signaling.

Microgravity (MG) leads to a decrease in osteogenic potential of human bone marrow-derived mesenchymal stem cells (hMSCs). In the present study, we used large gradient high magnetic field (LGHMF) produced by a superconducting magnet to model MG (LGHMF-MG) and analyzed the effects of LGHMF-MG on survival, cytoskeleton and osteogenic potential of hMSCs. Results showed that the LGHMF-MG treatment for 6 h disrupted the cytoskeleton of hMSCs, and the LGHMF-MG treatment for 24 h led to cell death. LGHMF-MG treatments for 6 h in early stages of osteogenic induction (the pre-treatment before osteogenic induction, the beginning-treatment in the beginning-stage of osteogenic induction and the middle-treatment in the middle-stage of osteogenic induction) resulted in suppression on osteogenesis of hMSCs. The suppression intensity was reduced gradually as the treatment stage of LGHMF-MG was postponed. The LGHMF-MG treatment for 6 h in the ending-stage of osteogenic induction (the ending-treatment) had no obvious effect on osteogenesis of hMSCs. These results indicated that LGHMF-MG should affect the initiation of osteogenesis. Finally, the possible mechanism for the inhibition effect of LGHMF-MG on osteogenesis of hMSCs is discussed.

AIM: To expand hematopoietic/progenitor stem cells (HS/PCs) from umbilical cord blood (UCB) and prepare the HS/PC product, and analyze preclinical transplantation and safety of HS/PC product. METHODS: Human bone marrow-derived mesenchymal stem cells (MSCs) were used as feeder cells to expand HS/PCs from UCB in a serum-free culture system. The proliferation potential of HS/PCs was analyzed. The expanded HS/PCs were suspended in the L-15 medium to prepare the HS/PC product. The contamination of bacteria, fungi and mycoplasmas, the infection of exogenous virus, the concentration of bacterial endotoxin, and the SCF residual in HS/PC product were determined. Finally, cells from the HS/PC product with or without bone marrow-derived mesenchymal stem cells (BM-MSCs) were transplanted into the irradiated NOD/SCID mice to determine the in vivo engraftment potential. RESULTS: After co-culture for 10 d, the total nuclear cells (TNCs) increased 125-fold, and CD34+ cells increased 43-fold. The granulocyte-macrophage colony- forming cells (GM-CFCs) and erythroid colony-forming cells (E-CFCs) increased 3.3- and 4.7-fold respectively. The expanded cells were collected and prepared as the expanded product of HS/PCs by re-suspending cells in L-15 medium. For preclinical safety, the HS/PC product was analysed for contamination by bacteria, fungi and mycoplasmas, the bacterial endotoxin concentration and the SCF content. The results showed that the HS/PC product contained no bacteria, fungi or mycoplasmas. The bacterial endotoxin concentration was less than the detection limit of 6 EU/mL, and residual SCF was 75 pg/mL. Based on clinical safety, the HS/PC product was qualified for clinical transplantation. Finally, the HS/PC product was transplanted the irradiated mice where it resulted in rapid engraftment of hematopoietic cells. CONCLUSION: HSPC product prepared from UCB in the serum-free culture system with hMSCs as feeder cells should be clinically safe and effective for clinical transplantation.

Mechanical stimuli can improve bone function by promoting the proliferation and differentiation of bone cells and osteoblasts. As precursors of osteoblasts, human mesenchymal stem cells (hMSCs) are sensitive to mechanical stimuli. In recent years, fluid shear stress (FSS) has been widely used as a method of mechanical stimulation in bone tissue engineering to induce the osteogenic differentiation of hMSCs. However, the mechanism of this differentiation is not completely clear. Several signaling pathways are involved in the mechanotransduction of hMSCs responding to FSS, such as MAPK, NO/cGMP/PKG and Ca(2+) signaling pathway. Here, we briefly review how hMSCs respond to fluid flow stimuli and focus on the signal molecules involved in this mechanotransduction.

Objectives: Previous studies have reported immortalization and tumorigenicity of human mesenchymal stem cells (hMSCs) transduced with exogenous human telomerase reverse transcriptase (hTERT). We also have established a line of hMSCs transduced with hTERT (hTERT–hMSCs) and we have cultured these cells for 290 population doublings (PDs) during which they demonstrated a large proliferation potential but with no tumorigenicity. The aim of this study was to investigate the protein expression profile of hTERT–hMSCs with two‐dimensional gel electrophoresis and peptide mass fingerprinting by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry, to be able to analyse the effects of exogenous hTERT on protein expression in hMSCs. Materials and methods: We generated proteome maps of primary hMSCs and hTERT–hMSCs at PD 95 and PD 275. Results: A total of 1543 ± 145 protein spots in gels of primary MSCs at PD 12, 1611 ± 186 protein spots in gels of hTERT–hMSCs at PD 95 and 1451 ± 126 protein spots in gels of hTERT–hMSCs at 275 PD were detected. One hundred of these were successfully identified, including 20 which were differentially expressed. Conclusions: The results suggest that sustaining levels of prohibitin and p53 expression along with differential expression of proteins in hTERT–hMSCs provide an insight into lack of transforming activity of hTERT–hMSCs during cell proliferation.

Human bone marrow mesenchymal stem cells (hMSCs) are promising candidates for cell therapy and tissue engineering. The life span of hMSCs during in vitro culture is limited. Human telomerase catalytic subunit (hTERT) gene transduction can prolong the life span of hMSCs and maintain their potential of osteogenic differentiation. We established a line of hMSCs transduced with exogenous hTERT (hTERT-hMSCs) and investigated its sustaining cellular properties in a long-term culture. This line of hTERT-hMSCs was cultured for 290 population doublings (PDs) without loss of contact inhibition. Under adipogenic, chondrogenic and osteogenic induction, hTERT-hMSCs at PD 95 and PD 275 could differentiate respectively into adipocytes, chondrocytes, and osteocytes. hTERT-hMSCs at these PDs showed no transforming activity through both in vitro assay of cell growth in soft agar and in vivo assay of tumorigenicity in NOD-SCID mice. Karyotype analyses showed no significant chromosomal abnormalities in hTERT-hMSCs at these PDs. These results suggested that the hTERT-hMSCs at lower population doubling levels (PDLs) should be considered as a cell model for studies of cellular senescence, differentiation and in vitro tissue engineering experiment because of its prolonged life span and normal cellular properties.

Microgravity (MG) results in a reduction in bone formation. Bone formation involves osteogenic differentiation from mesenchymal stem cells (hMSCs) in bone marrow. We modeled MG to determine its effects on osteogenesis of hMSCs and used activators or inhibitors of signaling factors to regulate osteogenic differentiation. Under osteogenic induction, MG reduced osteogenic differentiation of hMSCs and decreased the expression of osteoblast gene markers. The expression of Runx2 was also inhibited, whereas the expression of PPARγ2 increased. MG also decreased phosphorylation of ERK, but increased phosphorylation of p38MAPK. SB203580, a p38MAPK inhibitor, was able to inhibit the phosphorylation of p38MAPK, but did not reduce the expression of PPARγ2. Bone morphogenetic protein (BMP) increased the expression of Runx2. Fibroblast growth factor 2 (FGF2) increased the phosphorylation of ERK, but did not significantly increase the expression of osteoblast gene markers. The combination of BMP, FGF2 and SB203580 significantly reversed the effect of MG on osteogenic differentiation of hMSCs. Our results suggest that modeled MG inhibits the osteogenic differentiation and increases the adipogenic differentiation of hMSCs through different signaling pathways. Therefore, the effect of MG on the differentiation of hMSCs could be reversed by the mediation of signaling pathways.

This study is aimed at investigating the potentials of ex vivo expansion and pluri-differentiation of cryopreservation of adult human bone marrow mesenchymal stem cells (hMSCs) into chondrocytes, adipocytes and neurocytes. Cryopreserved hMSCs were resuscitated and cultured for 15 passages, and then induced into chondrocytes, adipocytes and neurocytes with corresponding induction medium. The induced cells were observed for morphological properties and detected for expressions of type II collagen, triglyceride or neuron-specific enolase and nestin. The result showed that the resuscitated cells could differentiate into chondrocytes after exposure to transforming growth factor beta(1) (TGF-beta(1)), insulin-like growth factor I (IGF-I) and vitamin C (V(C)), and uniformly changed morphologically from a spindle-like fibroblastic appearance to a polygonal shape in three weeks. The induced cells were heterochromatic to safranin O and expressed cartilage matrix-procollagenal (II) mRNA. The resuscitated cells cultured in induction medium consisting of dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin and IGF-I showed adipogenesis, and lipid vacuoles accumulation was detectable after 21 d. The resuscitated hMSCs were also induced into neurocytes and expressed nestin and neuron specific endolase (NSE) that were special surface markers associated with neural cells at different stage. This study suggested that the resuscitated hMSCs should be still a population of pluripotential cells and that it could be used for establishing an abundant hMSC reservoir for further experiment and treatment of various clinical diseases.

A novel indirect co-culture system was established to support ex vivo expansion of hematopoietic progenitors in umbilical cord blood (UCB) by using thrombopoietin (TPO)/Flt-3 ligand (FL)-transduced human-marrow-derived mesenchymal stem cells (tfhMSCs) as a feeder. UCB CD34+ cells were isolated and cultured by using five culture systems in serum-containing or serum-free medium. Suitable aliquots of cultured cells were taken to monitor cell production, clonogenic activity, and long-term culture-initiating culture (LTC-IC) output. Finally, the severe-combined immunodeficient mouse (SCID) repopulating cell (SRC) assay was performed to confirm the ability of the indirect co-cultured cells from the tfhMSCs system to reconstitute long-term hematopoiesis. Results showed significant differences in the number of total nucleated cells (TNCs) among the culture systems with respect to serum-containing medium or serum-free medium during 14-day culture. In addition, on day 14, the outputs of CD34+ cells, the colony-forming units (CFUs) in culture, and the CFUs in mixed colonies containing erythroid and myeloid cells and megakaryocytes in the tfhMSC indirect co-culture system were significantly enhanced. The LTC-IC assay demonstrated that the tfhMSCs indirect co-culture system had the strongest activity. The SCID-SRC assay confirmed the extensive ability of the expanded cells from the tfhMSCs indirect co-culture systems to reconstitute long-term hematopoiesis. Furthermore, polymerase chain reaction analysis demonstrated the presence of human hematopoietic cells in the bone marrow and peripheral blood cells of non-obese diabetic/SCID mice. Thus, hMSCs transduced with TPO/FL, in combination with additive cytokines, can effectively expand hematopoietic progenitors from UCB in vitro. The tfhMSC indirect co-culture system may therefore be a suitable system for ex vivo manipulation of primitive progenitor cells under non-contact culture conditions.

Microgravity decreases osteoblastic activity, induces actin microfilament disruption and inhibits the responsiveness of osteoblast to cytokines, but the mechanisms remains enigmatic. The F-actin cytoskeleton has previously been implicated in manifold changes of cell shape, function and signaling observed under microgravity. Here we investigate the involvement of microfilament in mediating the effects of microgravity and BMP2 induction on Cbfa1 activity. For this purpose we constructed a fluorescent reporter cell line (OSE-MG63) of Cbfa1 activity by stably transfecting MG63 cells with a reporter consisting of six tandem copies of OSE2 and a minimal mOG2 promoter upstream of enhanced green fluorescent protein (EGFP). The fluorescence intensity of OSE-MG63 showed responsiveness to bone-related cytokines (IGF-I, vitamin D3 and BMP2) and presented an accordant tendency with alkaline phosphatase (ALP) activity. Using OSE-MG63 reporter fluorescence, we performed a semi-quantitative analysis of Cbfa1 activity after treatment with simulated microgravity, microfilament-disrupting agent (cytochalasin B, CB), microfilament-stabilizing agent (Jasplakinolide, JAS) or any combination thereof. In parallel, ALP activity, DNA binding activity of Cbfa1 to OSE2 (ChIP), F-actin structure (immunofluorescence) and EGFP mRNA expression (RT-qPCR) were analyzed. Simulated microgravity inhibited Cbfa1 activity, affected the responsiveness of Cbfa1 to cytokine BMP2, and caused a thinning and dispersed distribution of microfilament. Under normal gravity, CB significantly attenuated BMP2 induction to Cbfa1 activity as well as DNA binding activity of Cbfa1 to OSE2. The addition of JAS reversed the inhibitory effects of microgravity on the responsiveness of Cbfa1 to BMP2. Our study demonstrates that disrupting the microfilament organization by CB or simulated microgravity attenuates the responsiveness of Cbfa1 to BMP2. A stabilization of the microfilament organization by JAS reverses this inhibition. Taken together, these results suggest that actin microfilament participates in BMP2's induction to Cbfa1 activity and that their disruption might be an important contributor to microgravity's inhibition on BMP2's osteogenic induction.

The thermo-responsivity of PNIPAAm [poly(N-isopropylcarylamide)]-grafted PDMS [poly(dimethylsiloxane)] surface is a property that could be feasibly used for detaching cells adhered on the surface. We used benzophenone-initiated photopolymerization to graft PNIPAAm on PDMS substrates to construct the PNIPAAm-grafted PDMS surface and this PDMS surface was highly thermo-responsive. hMSCs (human mesenchymal stem cells) were used to analyse the proliferation and multi-differentiation of stem cells on the PNIPAAm-grafted PDMS surface. The results showed that hMSCs could adhere on the PNIPAAm-grafted PDMS surface at 37°C and form cell colonies, and then become fibroblastic. The proliferation potential of hMSCs on the PNIPAAm-grafted PDMS surface was not significantly different from that on a plate surface coated with gelatin. However, as it proved easier to detach cells from the surface, by changing temperature, a higher viability of detached cells could be obtained with the PNIPAAm-grafted PDMS surface, using a temperature shift, compared with a gelatin-coated surface, where cells are detached by treatment with trypsin. hMSCs on the PNIPAAm-grafted PDMS surface were induced into osteoblasts, adipocytes and neurocytes under osteogenic medium, adipogenic medium and neurogenic medium respectively. The PNIPAAm-grafted PDMS surface was favourable for osteogenesis of hMSCs, although the potentials of adipogenesis and neurogenesis of hMSCs on the PNIPAAm-grafted PDMS surface were similar to those on the plate surface coated with gelatin. The above results demonstrate that the PNIPAAm-grafted PDMS surface not only kept the potentials of proliferation and multi-differentiation of hMSCs, but also increased the viability of hMSCs.

It has been verified that placenta contains multi-lineage mesenchymal stem cells (MSCs). We have used a time-gradient attachment method to isolate placenta-derived MSCs (PMSCs). The morphology, differentiation potential, immunogenicity and xenogenic reconstruction potential of these PMSCs were examined. The results showed that PMSCs isolated using the time-gradient attachment method showed higher potential of in vitro proliferation and multi-lineage differentiation. PMSCs isolated using the time-gradient attachment method showed a low immunogenicity. HLA-A gene fragment and no HLA-DR gene fragment were detected in PMSCs isolated using the time-gradient attachment method, and the mixed lymphocyte reaction (MLR) assay identified that these cells inhibited the proliferation of the allogeneic T-lymphocytes induced by PHA. The transplantation in calvaria of rats showed that PMSCs had the higher xenogenic reconstruction potential. Finally, the significance of PMSCs isolated using the time-gradient attachment method in experimental and clinical applications is discussed.