Human Fetal Mesenchymal Stem Cells Research Articles

Identification of candidate surface antigens for non-invasive prenatal diagnosis by comparative global gene expression on human fetal mesenchymal stem cells.

The authors wish to inform the reader that one image in Figure 3 was by mistake duplicated (OSMR LI 1 and BM 3) in the compilation of the figure. The inadvertent image display error does not change the results or the conclusions in the paper. 'Figure Presented'.

Mitochondrial DNA Hypomethylation Is a Biomarker Associated with Induced Senescence in Human Fetal Heart Mesenchymal Stem Cells.

Background. Fetal heart can regenerate to restore its normal anatomy and function in response to injury, but this regenerative capacity is lost within the first week of postnatal life. Although the specific molecular mechanisms remain to be defined, it is presumed that aging of cardiac stem or progenitor cells may contribute to the loss of regenerative potential. Methods. To study this aging-related dysfunction, we cultured mesenchymal stem cells (MSCs) from human fetal heart tissues. Senescence was induced by exposing cells to chronic oxidative stress/low serum. Mitochondrial DNA methylation was examined during the period of senescence. Results. Senescent MSCs exhibited flattened and enlarged morphology and were positive for the senescence-associated beta-galactosidase (SA-β-Gal). By scanning the entire mitochondrial genome, we found that four CpG islands were hypomethylated in close association with senescence in MSCs. The mitochondrial COX1 gene, which encodes the main subunit of the cytochrome c oxidase complex and contains the differentially methylated CpG island 4, was upregulated in MSCs in parallel with the onset of senescence. Knockdown of DNA methyltransferases (DNMT1, DNMT3a, and DNMT3B) also upregulated COX1 expression and induced cellular senescence in MSCs. Conclusions. This study demonstrates that mitochondrial CpG hypomethylation may serve as a critical biomarker associated with cellular senescence induced by chronic oxidative stress.

Counteracting bone fragility with human amniotic mesenchymal stem cells.

The impaired maturation of bone-forming osteoblasts results in reduced bone formation and subsequent bone weakening, which leads to a number of conditions such as osteogenesis imperfecta (OI). Transplantation of human fetal mesenchymal stem cells has been proposed as skeletal anabolic therapy to enhance bone formation, but the mechanisms underlying the contribution of the donor cells to bone health are poorly understood and require further elucidation. Here, we show that intraperitoneal injection of human amniotic mesenchymal stem cells (AFSCs) into a mouse model of OI (oim mice) reduced fracture susceptibility, increased bone strength, improved bone quality and micro-architecture, normalised bone remodelling and reduced TNFα and TGFβ sigalling. Donor cells engrafted into bones and differentiated into osteoblasts but importantly, also promoted endogenous osteogenesis and the maturation of resident osteoblasts. Together, these findings identify AFSC transplantation as a countermeasure to bone fragility. These data have wider implications for bone health and fracture reduction.

Fusion of Human Fetal Mesenchymal Stem Cells with "Degenerating" Cerebellar Neurons in Spinocerebellar Ataxia Type 1 Model Mice.

Mesenchymal stem cells (MSCs) migrate to damaged tissues, where they participate in tissue repair. Human fetal MSCs (hfMSCs), compared with adult MSCs, have higher proliferation rates, a greater differentiation capacity and longer telomeres with reduced senescence. Therefore, transplantation of quality controlled hfMSCs is a promising therapeutic intervention. Previous studies have shown that intravenous or intracortical injections of MSCs result in the emergence of binucleated cerebellar Purkinje cells (PCs) containing an MSC-derived marker protein in mice, thus suggesting a fusion event. However, transdifferentiation of MSCs into PCs or transfer of a marker protein from an MSC to a PC cannot be ruled out. In this study, we unequivocally demonstrated the fusion of hfMSCs with murine PCs through a tetracycline-regulated (Tet-off) system with or without a Cre-dependent genetic inversion switch (flip-excision; FLEx). In the FLEx-Tet system, we performed intra-cerebellar injection of viral vectors expressing tetracycline transactivator (tTA) and Cre recombinase into either non-symptomatic (4-week-old) or clearly symptomatic (6–8-month-old) spinocerebellar ataxia type 1 (SCA1) mice. Then, the mice received an injection of 50,000 genetically engineered hfMSCs that expressed GFP only in the presence of Cre recombinase and tTA. We observed a significant emergence of GFP-expressing PCs and interneurons in symptomatic, but not non-symptomatic, SCA1 mice 2 weeks after the MSC injection. These results, together with the results obtained using age-matched wild-type mice, led us to conclude that hfMSCs have the potential to preferentially fuse with degenerating PCs and interneurons but not with healthy neurons.

Human fetal mesenchymal stem cell secretome enhances bone consolidation in distraction osteogenesis.

BackgroundDistraction osteogenesis (DO) is one of the most dramatic reconstructive techniques for inducing bone regeneration, but it involves an undesirably long period for bone consolidation. Developing innovative approaches to enhance bone consolidation is a burning need. Human fetal mesenchymal stem cells (hFMSCs) have been shown to express more primitive developmental genes than those of human adult mesenchymal stem cells (hAMSCs), which is a preferable source for cell therapy and tissue regeneration. In the present study, we investigated the immunogenicity of using the human mesenchymal stem cell (MSC) secretome on rat cells, the effects of secretome on osteogenic differentiation of rat bone marrow-derived MSCs (rBMSCs), and the potential application of hFMSC secretome in promoting bone consolidation in a rat DO model.MethodsSecretome was collected from MSC culture and was used to treat rBMSCs. Following secretome treatment, cell proliferation, alkaline phosphatase staining, Alizarin Red S staining, and mRNA expression of osteogenic differentiation-related genes (including ALP, Runx2, OCN, OPN, and Osx) in the rBMSCs were checked, as well as mixed rat peripheral blood lymphocyte reaction. hFMSC secretome was injected locally into the regenerates from the end of lengthening every 3 days in the rat DO model, until termination. The regenerates were subject to weekly x-rays, micro-computed tomography (μCT) and mechanical testing examination. The bone quality was assessed by histology and immunohistochemistry examinations.ResultsCompared to the secretome from rBMSCs and hAMSCs, hFMSC secretome had the best osteogenic induction ability and low immunogenicity. hFMSC secretome with different doses showed no effect on cell viability. hFMSC secretome at the dose of 100 μg/μl could significantly increase the expression of alkaline phosphatase and all the osteogenic marker genes, as well as the amount of calcium deposits in the rBMSCs. Finally, the local application of hFMSC secretome in distraction regenerates in a rat DO model significantly improved bone consolidation according to the results of μCT, mechanical test, and histological and immunohistochemistry analysis.ConclusionsThe current study demonstrated that hFMSC secretome promotes osteogenesis of rBMSCs and bone consolidation during DO. hFMSC secretome may be a new therapeutic strategy to enhance bone consolidation in patients undergoing DO treatment.

Development and Characterization of Organic Electronic Scaffolds for Bone Tissue Engineering.

Bones have been shown to exhibit piezoelectric properties, generating electrical potential upon mechanical deformation and responding to electrical stimulation with the generation of mechanical stress. Thus, the effects of electrical stimulation on bone tissue engineering have been extensively studied. However, in bone regeneration applications, only few studies have focused on the use of electroactive 3D biodegradable scaffolds at the interphase with stem cells. Here a method is described to combine the bone regeneration capabilities of 3D-printed macroporous medical grade polycaprolactone (PCL) scaffolds with the electrical and electrochemical capabilities of the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT). PCL scaffolds have been highly effective in vivo as bone regeneration grafts, and PEDOT is a leading material in the field of organic bioelectronics, due to its stability, conformability, and biocompatibility. A protocol is reported for scaffolds functionalization with PEDOT, using vapor-phase polymerization, resulting in a conformal conducting layer. Scaffolds' porosity and mechanical stability, important for in vivo bone regeneration applications, are retained. Human fetal mesenchymal stem cells proliferation is assessed on the functionalized scaffolds, showing the cytocompatibility of the polymeric coating. Altogether, these results show the feasibility of the proposed approach to obtain electroactive scaffolds for electrical stimulation of stem cells for regenerative medicine.

Synthesis, molecular docking and biological evaluation of N,N-disubstituted 2-aminothiazolines as a new class of butyrylcholinesterase and carboxylesterase inhibitors

A series of 31 N,N-disubstituted 2-amino-5-halomethyl-2-thiazolines was designed, synthesized, and evaluated for inhibitory potential against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and carboxylesterase (CaE). The compounds did not inhibit AChE; the most active compounds inhibited BChE and CaE with IC50 values of 0.22–2.3μM. Pyridine-containing compounds were more selective toward BChE; compounds with the para-OMe substituent in one of the two dibenzyl fragments were more selective toward CaE. Iodinated derivatives were more effective BChE inhibitors than brominated ones, while there was no influence of halogen type on CaE inhibition. Inhibition kinetics for the 9 most active compounds indicated non-competitive inhibition of CaE and varied mechanisms (competitive, non-competitive, or mixed-type) for inhibition of BChE. Docking simulations predicted key binding interactions of compounds with BChE and CaE and revealed that the best docked positions in BChE were at the bottom of the gorge in close proximity to the catalytic residues in the active site. In contrast, the best binding positions for CaE were clustered rather far from the active site at the top of the gorge. Thus, the docking results provided insight into differences in kinetic mechanisms and inhibitor activities of the tested compounds. A cytotoxicity test using the MTT assay showed that within solubility limits (<30μM), none of the tested compounds significantly affected viability of human fetal mesenchymal stem cells. The results indicate that a new series of N,N-disubstituted 2-aminothiazolines could serve as BChE and CaE inhibitors for potential medicinal applications.

Hepatocytic Differentiation Potential of Human Fetal Liver Mesenchymal Stem Cells: In Vitro and In Vivo Evaluation.

In line with the search of effective stem cell population that would progress liver cell therapy and because the rate and differentiation potential of mesenchymal stem cells (MSC) decreases with age, the current study investigates the hepatogenic differentiation potential of human fetal liver MSCs (FL-MSCs). After isolation from 11-12 gestational weeks' human fetal livers, FL-MSCs were shown to express characteristic markers such as CD73, CD90, and CD146 and to display adipocytic and osteoblastic differentiation potential. Thereafter, we explored their hepatocytic differentiation potential using the hepatogenic protocol applied for adult human liver mesenchymal cells. FL-MSCs differentiated in this way displayed significant features of hepatocyte-like cells as demonstrated in vitro by the upregulated expression of specific hepatocytic markers and the induction of metabolic functions including CYP3A4 activity, indocyanine green uptake/release, and glucose 6-phosphatase activity. Following transplantation, naive and differentiated FL-MSC were engrafted into the hepatic parenchyma of newborn immunodeficient mice and differentiated in situ. Hence, FL-MSCs appeared to be interesting candidates to investigate the liver development at the mesenchymal compartment level. Standardization of their isolation, expansion, and differentiation may also support their use for liver cell-based therapy development.

Soluble Factors from Human Fetal Bone Marrow-Derived Mesenchymal Stem Cells: Preparation of Conditioned Medium and Its Effect on Tumor Cells.

Mesenchymal stem cells (MSCs) possess some unique features (inherent tumor tropism, anti-inflammatory and immunosuppressive properties) that are not commonly found in conventional anti-cancer agents. These cells are known to secrete a vast array of proteins including growth factors, cytokines, chemokines, extracellular matrix metalloproteinases, and their corresponding inhibitors which exhibit profound effects on the microenvironment. However, the lack of a uniform method for culturing MSCs and their paracrine factors has hindered our understanding of MSC biology. In this chapter, we describe methods for the isolation, in vivo expansion, and phenotypic characterization of MSCs. In addition, methods for the collection and concentration of conditioned medium from these MSCs are described. Using tumor cells that constitutively express fluorescence reporter proteins, the effect of conditioned medium on tumor cell viability can be easily tested in vitro.

Abstract 3204: Tissue engineered models of metastatic bone disease for the study of prostate cancer cell dormancy

Abstract Cellular dormancy refers to a state of quiescence during metastatic progression, in which disseminated tumor cells enter cell cycle arrest. Such dormant cells are thought to influence cancer drug sensitivity, and strategies have hence emerged to target dormant cells for novel therapeutic approaches. Mechanisms of cellular dormancy remain poorly understood, with current evidence suggesting a major role of the metastatic microenvironment in deciding cell fate. Research in this area, however, is hampered by the lack of appropriate experimental models of cancer cell dormancy. Here, we described the use of tissue engineering methods to generate an in vitro model of bone-metastatic disease. Briefly, human fetal mesenchymal stem cell (hfMSC) and human umbilical vein endothelial cell (HUVEC) were co-cultured to respectively represent the osteo-progenitor and vascular compartments, and subsequently induced to form a vascularized bone marrow model. Metastatic prostate cancer PC3 cells were then added to simulate metastatic dissemination and the co-culture was maintained for 28 days. Reporter genes were employed to facilitate live-monitoring of HUVEC and PC3 cells in the co-culture. Using this system, we observed single cell dormancy, particularly when osteogenic induction was performed. Additionally, PC3 cells were observed to exert osteo-modulatory effects on the engineered bone microenvironment. Immunostaining for cellular markers indicated low expression of proliferation marker Ki-67 and various mesenchymal markers, indicating absence of Epithelial-Mesenchymal Transition (EMT). Dormancy was functionally assessed by retrieving the PC3 cells and subsequently replating under normal culture conditions to rescue cellular proliferation. Finally, drug sensitivity assays demonstrated reduced susceptibility of dormant cells to conventional chemotherapeutic drugs. This in vitro model of prostate cancer cell dormancy in the engineered bone marrow may thus be useful for furthering our understanding of cellular dormancy, and may further provide a useful tool for drug testing. Citation Format: Yanwan Liao, Jun Jie Aaron Tan, Sahaana Tamilselvan, Jerry Chan, Ruowen Ge, Lui Shiong Lee, Mark Chong. Tissue engineered models of metastatic bone disease for the study of prostate cancer cell dormancy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3204. doi:10.1158/1538-7445.AM2015-3204

Combination of retinoic acid, dimethyl sulfoxide and 5-azacytidine promotes cardiac differentiation of human fetal liver-derived mesenchymal stem cells.

There are controversial reports about cardiac differentiation potential of mesenchymal stem cells (MSCs), and there is still no well-defined protocol for the induction of cardiac differentiation. The effects of retinoic acid (RA) and dimethyl sulfoxide (DMSO) on the proliferation and differentiation of human fetal liver-derived MSCs (HFMSCs) as well as the pluripotent state induced by 5-azacytidine (5-aza) in vitro were investigated. MSCs were isolated from fetal livers and cultured in accordance with previous reports. Cells were plated and were treated for 24 h by the combination of 5-aza, RA and DMSO in different doses. Different culture conditions were tested in our study, including temperature, oxygen content and medium. Three weeks later, cells were harvested for the certification of cardiac differentiation as well as the pluripotency, which indicated by cardiac markers and Oct4. It was found that the cardiac differentiation was only induced when HFMSCs were treated in the following conditions: in high-dose combination (5-aza 50 μM + RA 10(-1) μM + DMSO 1 %) in cardiac differentiation medium at 37 °C and 20 % O2. The results of immunohistochemistry and quantitative RT-PCR showed that about 40 % of the cells positively expressed Nkx2.5, desmin and cardiac troponin I, as well as Oct4. No beating cells were observed during the period. The combined treatment with RA, DMSO and 5-aza in high-dose could promote HFMSCs to differentiate into cardiomyocyte-like cells and possibly through the change of their pluripotent state.

598. Fusion of Mesenchymal Stem Cells Exclusively with “Degenerating” Cerebellar Neurons in Spinocerebellar Ataxia Type 1 Model Mice

Mesenchymal stem cells (MSCs) have the potential to migrate to damaged tissues where they participate in tissue repair. Previous studies suggest that intravenous or intracortical injections of MSCs results in the emergence of binucleated cerebellar Purkinje cells (PCs) containing a MSC-derived marker protein in mice suggestive of a fusion event. However, transdifferentiation of MSCs into PCs or transfer of a marker protein from a MSC to a PC cannot be completely ruled-out. Here we unequivocally showed a fusion of human fetal MSCs (hMSCs) with mouse PCs by combination of tetracycline-regulated system with Cre-dependent genetic inversion switch (flip-excision; FLEx). We performed intra-cerebellar injection of adeno-associated viral vectors expressing tetracycline transactivator (tTA) and Cre recombinase into either non-symptomatic (4 week-old) or clearly symptomatic (6 – 8 month-old) spinocerebellar ataxia type 1 (SCA1) mice. Two weeks after the treatment, the mice received injection of 50,000 geneticially-engineered hMSCs that expressed GFP only in the presence of Cre recombinase and tTA, which restricted GFP expression exclusively to cerebellar cells fused with hMSCs. We observed GFP-expressing PCs and interneurons in symptomatic, but not non-symptomatic, SCA1 mice as early as 2 weeks after the MSC injection. The frequency of GFP-expressing PCs did not increase when analysed at longer time points (≈23 weeks post hMSC grafting), suggesting that most hMSC fusion events occurred within 2 weeks of delivery. Using this model, we show conclusively that hMSC display a tendency to fuse with degenerating PC and interneurons, and not with normal neurons.

Methylation status of the FHIT gene in the transformed human mesenchymal F6 stem cell line.

The fragile histidine triad (FHIT) gene is known to be a tumor suppressor gene and the abnormal methylation of FHIT has been identified in leukemia and several solid tumors. The transformation of the tumor F6 cell line from human fetal mesenchymal stem cells (FMSCs) was first reported in a previous study that also identified the presence of a population of cancer stem cells in the F6 cell line. However, the existence of the epigenetic changes during the transformation process have yet to be elucidated. To confirm the role of the FHIT gene in the transformation process of FMSC, the expression level and methylation status of the FHIT gene was examined in F6 tumor cells and FMSCs. Additionally, the alteration in cell morphology, the cell cycle and apoptosis in F6 cells following 5-Aza-CdR treatment was assessed. It was found that the FHIT gene was expressed in FMSCs, but not in F6 cells. The methylation-specific PCR results demonstrated that the promoter methylation of FHIT genes existed in the F6 cell line. Subsequent to treatment with 5-Aza-CdR the expression of FHIT genes was restored in F6 cells. In addition, the morphology of F6 cells was altered, and the cell cycle was arrested in the G2 phase, with the initiation of apoptosis. Overall, the present findings demonstrated that the FHIT gene was methylated in F6 cells and demethylation treatment lead to changes in the biological characteristics, thereby promoting the apoptosis of F6 cells. FHIT gene methylation may be one of the molecular events involved in the development and transformation of FMSCs into F6 tumor cells.

Paracrine Factors of Human Fetal MSCs Inhibit Liver Cancer Growth Through Reduced Activation of IGF-1R/PI3K/Akt Signaling

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death in the world. The multikinase inhibitor sorafenib only demonstrated marginal improvement in overall survival for advanced disease prompted the search for alternative treatment options. Human mesenchymal stem cells (MSCs) have the ability to home to tumor cells. However, its functional roles on the tumor microenvironment remain controversial. Herein, we showed that conditioned media derived from human fetal MSC (CM-hfMSCs) expressed high level of the insulin growth factor binding proteins IGFBPs and can sequester free insulin-like growth factors (IGFs) to inhibit HCC cell proliferation. The inhibitory effect of IGFBPs on IGF signaling was further evident from the reduction of activated IGF-1R and PI3K/Akt, leading eventually to the induction of cell cycle arrest. We also demonstrated that CM-hfMSCs could enhance the therapeutic efficacy of sorafenib and sunitinib. To the best of our knowledge, this is the first report to show that CM-hfMSCs has a tumor-specific, antiproliferative effect that is not observed with normal human hepatocyte cells and patient-derived matched normal tissues. Our results thus suggest that CM-hfMSCs can provide a useful tool to design alternative/adjuvant treatment strategies for HCC, especially in related function to potentiate the effects of chemotherapeutic drugs.

The influence of human fetal mesenchymal stem cells on glioma cell proliferation. The consequence of cellular crosstalk

The effect of mesenchymal stem cells (MSCs) on the growth of various tumors is ambiguous. MSCs derived from different tissues stimulate growth of some tumor types and exert antitumor effects on the other. Several recent reports have shown that crosstalk between tumor cells and MSCs contribute to these effects. The aim of this work was to study the effects of MSCs derived from fetal tissues on the proliferative activity of glioma cells in conditions of prolonged co-cultivation. We have analyzed the proliferative activity of glioma cells exposed to conditioned medium (CM) from MSCs derived from fetal bone marrow (FetMSC) and fetal muscle (M-FetMSC) as well as to CM from co-cultivation of the fetal MSCs with U251MG glioma cells. As a comparison, the influence of CM from adult dermal fibroblasts (DFs) was examined in identical experiments. Using MTT assay, we have found that CM from both the fetal MSCs and adult DFs (without their co-culturing with glioma cells) had no effect on U251MG and A172 glioma cell proliferation. However, CM from early co-cultures (3-9 days) of U251MG cells with FetMSC or M-FetMSC exerted stimulatory effect on U251MG cell proliferation up to 2.3-fold increase, while CM obtained later from the same co-cultures (15-21 days) had inhibitory effect on the proliferation up to arrest of cell division. Analogous experiments with adult DFs have revealed a persistent stimulation of U251MG cell proliferative activity for all 21 days of co-culturing. Immunofluorescence analysis revealed a reduction in the expression of cell cycle protein cyclin D1 in U251MG cells after their treatment with CM taken from 21-days co-cultures of U251MG cells with FetMSC or M-FetMSC. In contrast, CM from 21-days co-cultures of U251MG cells with DFs did not decrease the expression of cyclin D1. These results show that fetal MSCs have dual effect on glioma cell proliferation. In spite of the earlier stimulatory effect on the proliferative activity, prominent inhibition of glioma cell proliferation was observed after three week co-culturing of glioma cells with fetal MSCs. This is the first report demonstrating reversion of tumor cell proliferative program during co-culturing with MSCs for a long time. These data suggest that CM obtained at different time points of cell co-culturing can be used in the modeling of prolonged cellular crosstalk.

Alteration of histone acetylation pattern during long-term serum-free culture conditions of human fetal placental mesenchymal stem cells.

Increasing evidence suggests that the mesenchymal stem cells (MSCs) derived from placenta of fetal origin (fPMSCs) are superior to MSCs of other sources for cell therapy. Since the initial number of isolated MSCs is limited, in vitro propagation is often required to reach sufficient numbers of cells for therapeutic applications, during which MSCs may undergo genetic and/or epigenetic alterations that subsequently increase the probability of spontaneous malignant transformation. Thus, factors that influence genomic and epigenetic stability of MSCs following long-term expansions need to be clarified before cultured MSCs are employed for clinical settings. To date, the genetic and epigenetic stability of fPMSCs after long-term in vitro expansion has not been fully investigated. In this report, alterations to histone acetylation and consequence on the expression pattern of fPMSCs following in vitro propagation under serum-free conditions were explored. The results show that fPMSCs maintain their MSC characteristics before they reached a senescent state. Furthermore, acetylation modification patterns were changed in fPMSCs along with gradually increased global histone deacetylase (HDAC) activity and expression of HDAC subtypes HDAC4, HDAC5 and HDAC6, as well as a down-regulated global histone H3/H4 acetylation during in vitro culturing. In line with the acetylation alterations, the expression of oncogenes Oct4, Sox2 and TERT were significantly decreased over the propagation period. Of note, the down-regulation of Oct4 was strongly associated with changes in acetylation. Intriguingly, telomere length in fPMSCs did not significantly change during the propagating process. These findings suggest that human fPMSCs may be a safe and reliable resource of MSCs and can be propagated under serum-free conditions with less risk of spontaneous malignancy, and warrants further validation in clinical settings.

5' isomiR variation is of functional and evolutionary importance.

We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3′ and/or 5′ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5′ differences and in support of this we report that a 5′ isomiR-9–1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5′ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes.

Directing the fate of human and mouse mesenchymal stem cells by hydroxyl-methyl mixed self-assembled monolayers with varying wettability.

Self-assembled monolayers (SAMs) of alkanethiols on gold have been employed as model substrates to investigate the effects of surface chemistry on cell behavior. However, few studies were dedicated to the substrates with a controlled wettability in studying stem cell fate. Here, mixed hydroxyl (-OH) and methyl (-CH3) terminated SAMs were prepared to form substrates with varying wettability, which were used to study the effects of wettability on the adhesion, spreading, proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) from human and mouse origins. The numbers of adhered human fetal MSCs (hMSCs) and mouse bone marrow MSCs (mMSCs) were maximized on -OH/-CH3 mixed SAMs with a water contact angle of 40~70° and 70~90°, respectively. Hydrophilic mixed SAMs with a water contact angle of 20~70° also promoted the spreading of both hMSCs and mMSCs. Both hMSCs and mMSCs proliferation was most favored on hydrophilic SAMs with a water contact angle around 70°. In addition, a moderate hydrophilic surface (with a contact angle of 40~90° for hMSCs and 70° for mMSCs) promoted osteogenic differentiation in the presence of biological stimuli. Hydrophilic mixed SAMs with a moderate wettability tended to promote the expression of αvβ1 integrin of MSCs, indicating that the tunable wettability of the mixed SAMs may guide osteogenesis through mediating the αvβ1 integrin signaling pathway. Our work can direct the design of biomaterials with controllable wettability to promote the adhesion, proliferation and differentiation of MSCs from different sources.

Human Fetal and Adult Bone Marrow-Derived Mesenchymal Stem Cells Use Different Signaling Pathways for the Initiation of Chondrogenesis

Cartilage injuries and osteoarthritis are leading causes of disability in developed countries. The regeneration of damaged articular cartilage using cell transplantation or tissue engineering holds much promise but requires the identification of an appropriate cell source with a high proliferative propensity and consistent chondrogenic capacity. Human fetal mesenchymal stem cells (MSCs) have been isolated from a range of perinatal tissues, including first-trimester bone marrow, and have demonstrated enhanced expansion and differentiation potential. However, their ability to form mature chondrocytes for use in cartilage tissue engineering has not been clearly established. Here, we compare the chondrogenic potential of human MSCs isolated from fetal and adult bone marrow and show distinct differences in their responsiveness to specific growth factors. Transforming growth factor beta 3 (TGFβ3) induced chondrogenesis in adult but not fetal MSCs. In contrast, bone morphogenetic protein 2 (BMP2) induced chondrogenesis in fetal but not adult MSCs. When fetal MSCs co-stimulated with BMP2 and TGFβ3 were used for cartilage tissue engineering, they generated tissue with type II collagen and proteoglycan content comparable to adult MSCs treated with TGFβ3 alone. Investigation of the TGFβ/BMP signaling pathway showed that TGFβ3 induced phosphorylation of SMAD3 in adult but not fetal MSCs. These findings demonstrate that the initiation of chondrogenesis is modulated by distinct signaling mechanisms in fetal and adult MSCs. This study establishes the feasibility of using fetal MSCs in cartilage repair applications and proposes their potential as an in vitro system for modeling chondrogenic differentiation and skeletal development studies.

GMP-grade human fetal liver-derived mesenchymal stem cells for clinical transplantation.

Stem cell therapy seems a promising avenue in regenerative medicine. Within various stem cells, mesenchymal stem cells have progressively used for cellular therapy. Because of the age-related decreasing in the frequency and differentiating capacity of adult MSCs, fetal tissues such as fetal liver, lung, pancreas, spleen, etc. have been introduced as an alternative source of MSCs for cellular therapy. On the other hand, using stem cells as advanced therapy medicinal products, must be performed in compliance with cGMP as a quality assurance system to ensure the safety, quality, and identity of cell products during translation from the basic stem cell sciences into clinical cell transplantation. In this chapter the authors have demonstrated the manufacturing of GMP-grade human fetal liver-derived mesenchymal stem cells.