Neural differentiation of umbilical cord mesenchymal stem cells by sub-sonic vibration

AimsAdult stem cells, such as umbilical cord-derived mesenchymal stem cells (UC-MSCs), have the potential to differentiate into various types of cells, including neurons. Research has shown that mechanical stimulation induces a response in MSCs, specifically, low and high intensity sub-sonic vibration (SSV) has been shown to facilitate wound healing. In this study, the effects of SSV were examined by assessing the proliferation and differentiation properties of MSCs.Main methodshUC-MSCs were isolated from Wharton's jelly, including the smooth muscle layer of the umbilical cord. During subculture, the cells were passaged every 5–6 days using nonhematopoietic stem cell media. To measure the effect of sonic vibration, SSV was applied to these cells continuously for 5 days.Key findingsIn this study, the morphology of hUC-MSCs was altered to resemble neurons by SSV. Further, the mRNA and protein levels of neuron-specific markers, including MAP2, NF-L, and NeuroD1, increased. In addition, other neural cell markers, such as GFAP and O4, were increased. These results suggest that hUC-MSCs differentiated into neural cells upon SSV nonselectively. In a mechanism study, the ERK level increased in a time-dependent manner upon SSV for 12 h.SignificanceThe results of this study suggest that SSV caused hUC-MSCs to differentiate into neural cells via ERK activation.     

人滑膜间充质干细胞与人脐带间充质干细胞的生物学性状比较

该文旨在比较人滑膜间充质干细胞(human synovial mesenchymal stem cells,hSMSCs)与人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)的生物学性状.流式细胞仪鉴定hSMSCs和hUC-MSCs.比较两种间...     

Differentiation of hUC-MSC into dopaminergic-like cells after transduction with hepatocyte growth factor

Parkinson’s disease (PD) is a common neurodegenerative condition causing significant disability and thus negatively impacting quality of life. The recent advent of stem cell-based therapy has heralded the prospect of a potential restorative treatment option for PD. In particular, mesenchymal stem cells derived from human umbilical cord (hUC-MSCs) have great potential for developing a therapeutic agent as such. Furthermore, hepatocyte growth factor (HGF), which shows mitogenic and morphogenetic activities in a variety of cells, including MSC, and may be implicated in the pathophysiology of PD. As such, HGF may represent a new therapeutic target for the disease. In this study, we successfully isolated and facilitated the transduction of an adenoviral vector expressing HGF (Ad-HGF) into isolated hUC-MSCs. Following transduction, the hUC-MSCs can differentiate into dopaminergic neuron-like cells secreting dopamine, tyrosine hydroxylase, and dopamine transporter. Our data suggest that hUC-MSCs have the ability to differentiate into dopaminergic neurons after transduction with Ad-HGF, providing encouraging evidence to further explore this approach to the treatment of PD.     

pp60src tyrosine kinase modulates P19 embryonal carcinoma cell fate by inhibiting neuronal but not epithelial differentiation

P19 embryonal carcinoma cells provide an in vitro model system to analyze the events involved in neural differentiation. These multipotential stem cells can be induced by retinoic acid (RA) to differentiate into neural cells. We have investigated the ability of several variant forms of the protein-tyrosine kinase (PTK) pp60src to modulate cell fate determination in this system. Normally, P19 cells are induced to differentiate along a neural lineage when allowed to form extensive cell-cell contacts in large multicellular aggregates during exposure to RA. Through analysis of markers of epithelial (keratin and desmosomal proteins) and neuronal (neurofilament) cells we have found that RA-induced P19 cells transiently express epithelial markers before neuronal differentiation. Under these inductive conditions, expression of pp60v-src or expression of the neuronal variant pp60c-src+ inhibited neuronal differentiation, and resulted in maintained expression of an epithelial phenotype. Morphological analysis showed that expression of pp60src PTKs results in decreased cell-cell adhesion during the critical cell aggregation stage of the neural differentiation procedure. The effects of pp60v-src on cell fate and cell-cell adhesion could be mimicked by direct modulation of Ca+(+)-dependent cell-cell contact during RA induction of normal P19 cells. We conclude that the neural lineage of P19 cells includes an early epithelial intermediate and suggest that tyrosine phosphorylation can modulate cell fate determination during an early cell-cell adhesion-dependent event in neurogenesis.     

Renoprotective Effect of Human Umbilical Cord–Derived Mesenchymal Stem Cells in Immunodeficient Mice Suffering from Acute Kidney Injury

It is unknown whether human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) can improve the renal function of patients suffering from acute kidney injury. Moreover, before beginning clinical trials, it is necessary to investigate this renoprotective effect of hUC-MSCs in a xenogeneic model of acute kidney injury. However, no previous studies have examined the application of hUC-MSCs to immunodeficient mice suffering from acute kidney injury. The objectives of this study were to examine whether hUC-MSCs could improve renal function in nonobese diabetic-severe combined immune deficiency (NOD-SCID) mice suffering from acute kidney injury, and to investigate the mechanism(s) for hUC-MSCs to improve renal function in this xenogeneic model. Early (3 hr) and late (12 hr) administrations of hUC-MSCs (10⁶ cells) were performed via the external jugular vein into NOD-SCID mice suffering from either folic acid (FA) (250 mg/kg body weight) or vehicle. The results showed that early administration of hUC-MSCs improved the renal function of NOD-SCID mice suffering from FA-induced acute kidney injury, as evidenced by decreased serum urea nitrogen and serum creatinine levels, as well as a reduced tubular injury score. The beneficial effects of hUC-MSCs were through reducing apoptosis and promoting proliferation of renal tubular cells. These benefits were independent of inflammatory cytokine effects and transdifferentiation. Furthermore, this study is the first one to show that the reduced apoptosis of renal tubular cells by hUC-MSCs in this xenogeneic model is mediated through the mitochondrial pathway, and through the increase of Akt phosphorylation.     

Differentiation of Human Umbilical Cord Mesenchymal Stem Cells into Prostate-Like Epithelial Cells In Vivo

Although human umbilical cord mesenchymal stem cells (hUC-MSCs) have been identified as a new source of MSCs for potential application in regenerative medicine, their full potential of differentiation has not been determined. In particular, whether they have the capability to differentiate into epithelial cells of endodermal origin such as the prostate epithelial cells is unknown. Here we report that when hUC-MSCs were combined with rat urogenital sinus stromal cells (rUGSSs) and transplanted into the renal capsule in vivo, they could differentiate into prostate epithelial-like cells that could be verified by prostate epithelial cell-specific markers including the prostate specific antigen. The prostatic glandular structures formed in vivo displayed similar cellular architecture with lumens and branching features as seen for a normal prostate. In addition, the human origin of the hUC-MSCs was confirmed by immunocytochemistry for human nuclear antigen. These findings together indicate that hUC-MSCs have the capability to differentiate into epithelial-like cells that are normally derived from the endoderm, implicating their potential applications in tissue repair and regeneration of many endoderm-derived internal organs.     

Nodal-dependent Cripto signaling promotes cardiomyogenesis and redirects the neural fate of embryonic stem cells

The molecular mechanisms controlling inductive events leading to the specification and terminal differentiation of cardiomyocytes are still largely unknown. We have investigated the role of Cripto, an EGF-CFC factor, in the earliest stages of cardiomyogenesis. We find that both the timing of initiation and the duration of Cripto signaling are crucial for priming differentiation of embryonic stem (ES) cells into cardiomyocytes, indicating that Cripto acts early to determine the cardiac fate. Furthermore, we show that failure to activate Cripto signaling in this early window of time results in a direct conversion of ES cells into a neural fate. Moreover, the induction of Cripto activates the Smad2 pathway, and overexpression of activated forms of type I receptor ActRIB compensates for the lack of Cripto signaling in promoting cardiomyogenesis. Finally, we show that Nodal antagonists inhibit Cripto-regulated cardiomyocyte induction and differentiation in ES cells. All together our findings provide evidence for a novel role of the Nodal/Cripto/Alk4 pathway in this process.     

An early requirement for FGF signalling in the acquisition of neural cell fate in the chick embryo

BACKGROUND: In Xenopus embryos, fibroblast growth factors (FGFs) and secreted inhibitors of bone morphogenetic protein (BMP)-mediated signalling have been implicated in neural induction. The precise roles, if any, that these factors play in neural induction in amniotes remains to be established. RESULTS: To monitor the initial steps of neural induction in the chick embryo, we developed an in vitro assay of neural differentiation in epiblast cells. Using this assay, we found evidence that neural cell fate is specified in utero, before the generation of the primitive streak or Hensen's node. Early epiblast cells expressed both Bmp4 and Bmp7, but the expression of both genes was downregulated as cells acquired neural fate. During prestreak and gastrula stages, exposure of epiblast cells to BMP4 activity in vitro was sufficient to block the acquisition of neural fate and to promote the generation of epidermal cells. Fgf3 was also found to be expressed in the early epiblast, and ongoing FGF signalling in epiblast cells was required for acquisition of neural fate and for the suppression of Bmp4 and Bmp7 expression. CONCLUSIONS: The onset of neural differentiation in the chick embryo occurs in utero, before the generation of Hensen's node. Fgf3, Bmp4 and Bmp7 are each expressed in prospective neural cells, and FGF signalling appears to be required for the repression of Bmp expression and for the acquisition of neural fate. Subsequent exposure of epiblast cells to BMPs, however, can prevent the generation of neural tissue and induce cells of epidermal character.     

Mesenchymal stem cells alleviate experimental immune-mediated liver injury via chitinase 3-like protein 1-mediated T cell suppression

Liver diseases with different pathogenesis share common pathways of immune-mediated injury. Chitinase-3-like protein 1 (CHI3L1) was induced in both acute and chronic liver injuries, and recent studies reported that it possesses an immunosuppressive ability. CHI3L1 was also expressed in mesenchymal stem cells (MSCs), thus we investigates the role of CHI3L1 in MSC-based therapy for immune-mediated liver injury here. We found that CHI3L1 was highly expressed in human umbilical cord MSCs (hUC-MSCs). Downregulating CHI3L1 mitigated the ability of hUC-MSCs to inhibit T cell activation, proliferation and inflammatory cytokine secretion in vitro. Using Concanavalin A (Con A)-induced liver injury mouse model, we found that silencing CHI3L1 significantly abrogated the hUC-MSCs-mediated alleviation of liver injury, accompanying by weakened suppressive effects on infiltration and activation of hepatic T cells, and secretion of pro-inflammatory cytokines. In addition, recombinant CHI3L1 (rCHI3L1) administration inhibited the proliferation and function of activated T cells, and alleviated the Con A-induced liver injury in mice. Mechanistically, gene set enrichment analysis showed that JAK/STAT signalling pathway was one of the most significantly enriched gene pathways in T cells co-cultured with hUC-MSCs with CHI3L1 knockdown, and further study revealed that CHI3L1 secreted by hUC-MSCs inhibited the STAT1/3 signalling in T cells by upregulating peroxisome proliferator-activated receptor δ (PPARδ). Collectively, our data showed that CHI3L1 was a novel MSC-secreted immunosuppressive factor and provided new insights into therapeutic treatment of immune-mediated liver injury.Subject terms: T cells, Mesenchymal stem cells, Autoimmune hepatitis     

Effects of chlorogenic acid,caffeine, and coffee on behavioral and biochemical parameters of diabetic rats

Mesenchymal stem cells (MSCs) represent a potential therapeutic target for glioma. We determined the molecular mechanism of inhibitory effect of human umbilical cord-derived MSCs (hUC-MSCs) on the growth of C6 glioma cells. We demonstrated that hUC-MSCs inhibited C6 cell growth and modulated the cell cycle to G0/G1 phase. The expression of β-catenin and c-Myc was downregulated in C6 cells by conditioned media from hUC-MSCs, and the levels of secreted DKK1 were positively correlated with concentrations of hUCMSCs-CM. The inhibitory effect of hUC-MSCs on C6 cell proliferation was enhanced as the concentration of DKK1 in hUCMSCs-CM increased. When DKK1 was neutralized by anti-DKK1 antibody, the inhibitory effect of hUC-MSCs on C6 cells was attenuated. Furthermore, we found that conditioned media from hUC-MSCs transfection with siRNA targeting DKK1 mRNA or pEGFPN1-DKK1 plasmid lost or enhanced the abilities to regulate the Wnt signaling in C6 cells. Therefore, hUC-MSCs inhibited C6 glioma cell growth via secreting DKK1, an inhibitor of Wnt pathway, may represent a novel therapeutic strategy for malignant glioma.     

WNT1 and WNT3a promote expansion of melanocytes through distinct modes of action

Summary WNT1 and WNT3a have been described as having redundant roles in promoting the development of neural crest-derived melanocytes (NC-Ms). We used cell lineage restricted retroviral infections to examine the effects of WNT signaling on defined cell types in neural crest cultures. RCAS retroviral infections were targeted to melanoblasts (NC-M precursor cells) derived from transgenic mice that express the virus receptor, TVA, under the control of a melanoblast promoter (DCT). As expected, over 90% of DCT-TVA+ cells expressed early melanoblast markers MITF and KIT. However, by following the fate of infected cells in standard culture conditions, we find that only 5% of descendents were NC-Ms. The majority of the descendents were not NC-Ms, but expressed smooth muscle cell markers, demonstrating that mammalian melanoblasts are not committed to the NC-M lineage. RCAS infection of DCT-TVA+ cells demonstrated that overexpression of canonical WNT signaling genes (betaCAT, WNT3a or WNT1) can increase NC-M numbers in an endothelin dependent manner. However, WNT1 and WNT3a have different modes of action with respect to melanoblast fate. Intrinsic over-expression of betaCAT or WNT3a can increase NC-M numbers by biasing the fate of DCT-TVA+ cells to NC-Ms. In contrast, the DCT-TVA+ melanoblasts cannot respond to WNT1 signaling and do not alter their fate towards NC-M. Instead, WNT1 only increases NC-M numbers through paracrine signaling on melanoblast precursors to increase the numbers of neural crest cells that become NC-Ms.     

Effective naked plasmid DNA delivery into stem cells by microextrusion-based transient-transfection system for in situ cardiac repair

Background aims. Combining the use of transfection reagents and physical methods can markedly improve the efficiency of gene delivery; however, such methods often cause cell damage. Additionally, naked plasmids without any vector or physical stimulation are difficult to deliver into stem cells. In this study, we demonstrate a simple and rapid method to simultaneously facilitate efficient in situ naked gene delivery and form a bioactive hydrogel scaffold. Methods. Transfecting naked GATA binding protein 4 (GATA4) plasmids into human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) by co-extruding naked plasmids and hUC-MSCs with a biomimetic and negatively charged water-based biodegradable thermo-responsive polyurethane (PU) hydrogel through a microextrusion-based transient-transfection system can upregulate the other cardiac marker genes. Results. The PU hydrogels with optimized physicochemical properties (such as hard-soft segment composition, size, hardness and thermal gelation) induced GATA4-transfected hUC-MSCs to express the cardiac marker proteins and then differentiated into cardiomyocyte-like cells in 15 days. We further demonstrated that GATA4-transfected hUC-MSCs in PU hydrogel were capable of in situ revival of heart function in zebrafish in 30 days. Conclusions. Our results suggest that hUC-MSCs and naked plasmids encapsulated in PU hydrogels might represent a new strategy for in situ tissue therapy using the microextrusion-based transient-transfection system described here. This transfection system is simple, effective and safer than conventional technologies.     

Inhibition of glycogen synthase kinase-3 enhances the differentiation and reduces the proliferation of adult human olfactory epithelium neural precursors

The olfactory epithelium (OE) contains neural precursor cells which can be easily harvested from a minimally invasive nasal biopsy, making them a valuable cell source to study human neural cell lineages in health and disease. Glycogen synthase kinase-3 (GSK-3) has been implicated in the etiology and treatment of neuropsychiatric disorders and also in the regulation of murine neural precursor cell fate in vitro and in vivo. In this study, we examined the impact of decreased GSK-3 activity on the fate of adult human OE neural precursors in vitro. GSK-3 inhibition was achieved using ATP-competitive (6-bromoindirubin-3′-oxime and CHIR99021) or substrate-competitive (TAT-eIF2B) inhibitors to eliminate potential confounding effects on cell fate due to off-target kinase inhibition. GSK-3 inhibitors decreased the number of neural precursor cells in OE cell cultures through a reduction in proliferation. Decreased proliferation was not associated with a reduction in cell survival but was accompanied by a reduction in nestin expression and a substantial increase in the expression of the neuronal differentiation markers MAP1B and neurofilament (NF-M) after 10 days in culture. Taken together, these results suggest that GSK-3 inhibition promotes the early stages of neuronal differentiation in cultures of adult human neural precursors and provide insights into the mechanisms by which alterations in GSK-3 signaling affect adult human neurogenesis, a cellular process strongly suspected to play a role in the etiology of neuropsychiatric disorders.     

Cardiovascular malformations with normal smooth muscle differentiation in neural crest-specific type II TGFbeta receptor (Tgfbr2) mutant mice

Previous studies have demonstrated that TGFbeta induces a smooth muscle fate in primary neural crest cells in culture. By crossing a conditional allele of the type II TGFbeta receptor with the neural crest-specific Wnt1cre transgene, we have addressed the in vivo requirement for TGFbeta signaling in smooth muscle specification and differentiation. We find that elimination of the TGFbeta receptor does not alter neural crest cell specification to a smooth muscle fate in the cranial or cardiac domains, and that a smooth muscle fate is not realized by trunk neural crest cells in either control or mutant embryos. Instead, mutant embryos exhibit with complete penetrance two very specific and mechanistically distinct cardiovascular malformations--persistent truncus arteriosus (PTA) and interrupted aortic arch (IAA-B). Pharyngeal organ defects such as those seen in models of DiGeorge syndrome were not observed, arguing against an early perturbation of the cardiac neural crest cell lineage. We infer that TGFbeta is an essential morphogenic signal for the neural crest cell lineage in specific aspects of cardiovascular development, although one that is not required for smooth muscle differentiation.     

Temporally controlled modulation of FGF/ERK signaling directs midbrain dopaminergic neural progenitor fate in mouse and human pluripotent stem cells

Effective induction of midbrain-specific dopamine (mDA) neurons from stem cells is fundamental for realizing their potential in biomedical applications relevant to Parkinson's disease. During early development, the Otx2-positive neural tissues are patterned anterior-posteriorly to form the forebrain and midbrain under the influence of extracellular signaling such as FGF and Wnt. In the mesencephalon, sonic hedgehog (Shh) specifies a ventral progenitor fate in the floor plate region that later gives rise to mDA neurons. In this study, we systematically investigated the temporal actions of FGF signaling in mDA neuron fate specification of mouse and human pluripotent stem cells and mouse induced pluripotent stem cells. We show that a brief blockade of FGF signaling on exit of the lineage-primed epiblast pluripotent state initiates an early induction of Lmx1a and Foxa2 in nascent neural progenitors. In addition to inducing ventral midbrain characteristics, the FGF signaling blockade during neural induction also directs a midbrain fate in the anterior-posterior axis by suppressing caudalization as well as forebrain induction, leading to the maintenance of midbrain Otx2. Following a period of endogenous FGF signaling, subsequent enhancement of FGF signaling by Fgf8, in combination with Shh, promotes mDA neurogenesis and restricts alternative fates. Thus, a stepwise control of FGF signaling during distinct stages of stem cell neural fate conversion is crucial for reliable and highly efficient production of functional, authentic midbrain-specific dopaminergic neurons. Importantly, we provide evidence that this novel, small-molecule-based strategy applies to both mouse and human pluripotent stem cells.     

Long-term cultured mesenchymal stem cells frequently develop genomic mutations but do not undergo malignant transformation

Cultured human umbilical cord mesenchymal stem cells (hUC-MSCs) are being tested in several clinical trials and encouraging outcomes have been observed. To determine whether in vitro expansion influences the genomic stability of hUC-MSCs, we maintained nine hUC-MSC clones in long-term culture and comparatively analyzed them at early and late passages. All of the clones senesced in culture, exhibiting decreased telomerase activity and shortened telomeres. Two clones showed no DNA copy number variations (CNVs) at passage 30 (P30). Seven clones had ≥1 CNVs at P30 compared with P3, and one of these clones appeared trisomic chromosome 10 at the late passage. No tumor developed in immunodeficient mice injected with hUC-MSCs, regardless of whether the cells had CNVs at the late passage. mRNA-Seq analysis indicated that pathways of cell cycle control and DNA damage response were downregulated during in vitro culture in hUC-MSC clones that showed genomic instability, but the same pathways were upregulated in the clones with good genomic stability. These results demonstrated that hUC-MSCs can be cultured for many passages and attain a large number of cells, but most of the cultured hUC-MSCs develop genomic alterations. Although hUC-MSCs with genomic alterations do not undergo malignant transformation, periodic genomic monitoring and donor management focusing on genomic stability are recommended before these cells are used for clinical applications.     

Xbra3 elicits the production of neural tissue by a non-BMP-dependent mechanism in Xenopus sp

Ectoderm cells in animal caps from Xenopus embryos develop to form either epidermis or neural tissue depending upon their receipt of intercellular signals. To date, several secreted neural inducers have been identified which act through the local inhibition of bone morphogenetic protein (BMP) signaling, preventing differentiation to epidermis and resulting in adoption of neural fate. In this work, we have exploited an interspecies animal cap assay, which enables detection of the effects of signaling molecules produced by cells of one animal cap and influencing development in a second cap cultured in close apposition in a Holtfreter combination. We show that expression of the T-box protein, Xbra3, in one cap causes the production of a factor, which causes adoption of neural fate by cells of the other animal cap. The action of this factor is not inhibited by the over-expression of BMP in cells of the responding animal cap, or by the inhibition of Wnt signaling. These findings suggest the existence of a secreted signaling molecule that is able to induce ectodermal cells to adopt neural fate by a mechanism independent of the inhibition of the BMP or Wnt signaling pathways.