In vitro differentiation of unrestricted somatic stem cells into functional hepatic‐like cells displaying a hepatocyte‐like glucose metabolism

The hepatic‐like phenotype resulting from in vitro differentiation of unrestricted somatic stem cells (USSC) derived from human umbilical cord blood (CB) was analyzed with regard to functional and metabolic aspects. USSC can be differentiated into cells of all three germ layers in vitro and in vivo and, although they share many features with mesenchymal stroma cells (MSC), can be distinguished from these by their expression of DLK1 as well as a restricted adipogenic differentiation potential. For the differentiation procedure described herein, a novel three‐stage differentiation protocol resembling embryonic developmental processes of hepatic endoderm was applied. Hepatic pre‐induction was performed by activinA and FGF4 resulting in enhanced SOX17 and FOXA2 expression. Further differentiation was achieved sequentially by retinoic acid, FGF4, HGF, EGF, and OSM resulting in a hepatic endodermal identity, characterized by the expression of AFP and HNF1α. Thereafter, expression of G6PC, ARG1, FBP1, and HNF4α was observed, thus indicating progressive differentiation. Functional studies concerning albumin secretion, urea formation, and cytochrome‐p450‐3A4 (CYP3A4) enzyme activity confirmed the hepatic‐like phenotype. In order to characterize the differentiated cells at a metabolic level, USSC were incubated with [1‐¹³C]glucose. By tracing the fate of the molecule's label via isotopomer analysis using ¹³C NMR spectroscopy, formation of both glycogen and some gluconeogenetic activity could be observed providing evidence of a hepatocyte‐like glucose metabolism in differentiated USSC. In conclusion, the results of the present study indicate that USSC represent a stem cell source with a substantial hepatic differentiation capacity which hold the potential for clinical applications. J. Cell. Physiol. 225: 545–554, 2010. © 2010 Wiley‐Liss, Inc.     

Germ‐like cell differentiation from induced pluripotent stem cells (iPSCs)

Historically, our understanding of molecular genetic aspects of germ cell development has been limited. Recently, results demonstrated that the derivation of pluripotent stem cells may provide the necessary genetic system to study germ cell development. Here, we characterized an induced pluripotent stem cell (iPSC) line, which can spontaneously differentiate into embryonic bodies (EBs) after 3 days of suspension culture, expressing specific markers of three germ layers. Then, we induced the iPSCs to differentiate into germ cells by culturing adherent EBs in retinoic acid (RA) and porcine follicular fluid (PFF) differentiation medium or seminiferous tubule transplantation. Our results indicated that RA and PFF were beneficial for the derivation of germ cells and oocyte‐like cells from iPSCs, and iPSCs transplantation could make a contribution to repairing the testis of infertile mice. Our study offers an approach for further study on the development and the differentiation of germ cells derived from iPSCs. Copyright © 2012 John Wiley & Sons, Ltd.     

MIR‐34c regulates mouse embryonic stem cells differentiation into male germ‐like cells through RARg

Embryonic stem cells (ESCs) have the capacity to differentiate into nearly all sorts of cell types, including germ cells, which were regarded as one type of highly specialized cells in mammals, taking the responsibility of transferring genetic materials to the next generation. Studies on induction differentiation of murine embryonic stem cells (mESCs) into male germ cells, but with a low efficiency, basic reason is that the regulation mechanism of germ cell development in mammals is still unclear. miRNA might play an important role in spermatogenesis in mammals. In this study, several miRNAs, which might be related to spermatogenesis, were initially selected and detected in the mouse tissues by semi‐polymerase chain reaction (PCR) and quantitative real time (qRT)‐PCR to find a testis‐specific miRNA. To study its effect on mESCs differentiation into male germ cells, miR‐34c mimics were synthesized and pri‐miR‐34c‐GFP plasmid was constructed, transfected into mESCs and combined with retinoic acid induction. The effects of miR‐34c were analysed by morphology, alkaline phosphatase staining, qRT‐PCR_and immunofluorescent staining. The results showed that miR‐34c promoted mESCs differentiation into male germ‐like cells, to some extent. Then miR‐34c targeted genes were predicted by bioinformatics; Retinoic acid receptor gamma (RARg) was selected, and two dual‐luciferase reporter vectors contained the normal and mutated 3′untranslated region of RARg were constructed, respectively. By miRNA mimics and vector co‐transfection experiment, the predicted target gene‐RARg was confirmed. In conclusion, we found a mammalian male germ cell specific miRNA—miR‐34c, and it might be pivotal in mESCs differentiation into male germ cells through its target—RARg. Copyright © 2012 John Wiley & Sons, Ltd.     

Calcium: A novel and efficient inducer of differentiation of adipose‐derived stem cells into neuron‐like cells

This study comparatively investigated the effectiveness of calcium and other well‐known inducers such as isobutylmethylxanthine (IBMX) and insulin in differentiating human adipose‐derived stem cells (ADSCs) into neuronal‐like cells. ADSCs were immunophenotyped and differentiated into neuron‐like cells with different combinations of calcium, IBMX, and insulin. Calcium mobilization across the membrane was determined. Differentiated cells were characterized by cell cycle profiling, staining of Nissl bodies, detecting the gene expression level of markers such as neuronal nuclear antigen (NeuN), microtubule associated protein 2 (MAP2), neuron‐specific enolase (NSE), doublecortin, synapsin I, glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP) by quantitative real‐time polymerase chain reaction (quantitative real‐time polymerase chain reaction (qRT‐PCR) and protein level by the immunofluorescence technique. Treatment with Ca + IBMX + Ins induced neuronal appearance and projection of neurite‐like processes in the cells, accompanied with inhibition of proliferation and halt in the cell cycle. A significantly higher expression of MBP, GFAP, NeuN, NSE, synapsin 1, doublecortin, and MAP2 was detected in differentiated cells, confirming the advantages of Ca + IBMX + Ins to the other combinations of inducers. Here, we showed an efficient protocol for neuronal differentiation of ADSCs, and calcium fostered differentiation by augmenting the number of neuron‐like cells and instantaneous increase in the expression of neuronal markers.     

Reversine promotes porcine muscle derived stem cells (PMDSCs) differentiation into female germ‐like cells

A small molecular chemical‐Reversine has been shown to promote cell reprogramming and induce dedifferentiation of multiple terminally differentiated mesodermal origin cells, and then differentiate into other cell types within mesodermal lineages as well as neuroectodermal. However, the possibilities of these cells to give rise to germ cell lineages have not been examined. The objective of the current study was to detect the effect of Reversine on PMDSCs differentiation into germ cells. PMDSCs from fetal porcine skeletal muscle and their potential of differentiation into germ cells in vitro were investigated. The phenotype, proliferation potential, characteristic markers of the first adhesion cells (pp1), and the purified 2 times cells (pp3) were analyzed by growth curve, FACS, and RT‐PCR, respectively. Then, the purified cells were induced with 10% or 20% bovine follicular fluid (FF), the results showed that some of the induced pp3 cells were similar as porcine oocyte, and expressed germ cell and oocyte markers analyzed by semi‐quantitative RT‐PCR and immunofluorescent staining. Reversine clearly increased the potentiality of PMDSCs differentiation into large round germ‐like cells in FF induction medium analyzed by morpholgogy, QRT‐PCR and immunofluoresce. The BrdU labeled PMDSCs might differentiate into female germ‐like cells in recipient's kidney capsule, which were positive for germ cell and meiotic markers (Dazl, Vasa, Figla, Stra8, Scp3) and oocyte markers (Zp2, Zp3). These findings provided an efficient model to study the mechanism of cell proliferation and germ cell differentiation in livestock promoted by Reversine. J. Cell. Biochem. 113: 3629–3642, 2012. © 2012 Wiley Periodicals, Inc.     

Dopaminergic‐like cells from epigenetically reprogrammed mesenchymal stem cells

A number of recent studies have examined the ability of stem cells derived from different sources to differentiate into dopamine‐producing cells and ameliorate behavioural deficits in Parkinsonian models. Recently, using the approach of cell reprogramming by small cell‐permeable biological active compounds that involved in the regulation of chromatin structure and function, and interfere with specific cell signalling pathways that promote neural differentiation we have been able to generate neural‐like cells from human bone marrow (BM)‐derived MSCs (hMSCs). Neurally induced hMSCs (NI‐hMSCs) exhibited several neural properties and exerted beneficial therapeutic effect on tissue preservation and locomotor recovery in spinal cord injured rats. In this study, we aimed to determine whether hMSCs neuralized by this approach can generate dopaminergic (DA) neurons. Immunocytochemisty studies showed that approximately 50–60% of NI‐hMSCs expressed early and late dopaminergic marker such as Nurr‐1 and TH that was confirmed by Western blot. ELISA studies showed that NI‐hMSCs also secreted neurotrophins and dopamine. Hypoxia preconditioning prior to neural induction increased hMSCs proliferation, viability, expression TH and the secretion level of dopamine induced by ATP. Taken together, these studies demonstrated that hMSCs neurally modified by this original approach can be differentiated towards DA‐like neurons.