Surface expression of CXCR4 in unrestricted somatic stem cells and its regulation by growth factors

Umbilical cord blood‐derived USSCs (unrestricted somatic stem cells) have recently been considered as a potential source for stem cell therapy and transplantation due to their characteristics such as easy accessibility, low immunogenicity, self‐renewing and multilineage differentiation potential. Stem cell homing is a key factor in successful transplantation, which is regulated by CXCR4 in stem cells. In this study, we evaluated the expression of CXCR4 in USSCs different passages. Moreover, the effect of VEGF (vascular endothelial growth factor) and IGF‐1 (insulin‐like growth factor 1) on its expression was assessed. It was shown that the expression of CXCR4 in USSCs decreased with the increase in passage number. It was also revealed that VEGF increased surface expression and mRNA level of CXCR4 in USSCs, while IGF‐1 decreased its expression. When VEGF and IGF‐1 were administered simultaneously, CXCR4 expression was increased, but the expression level was less than VEGF alone. Finally, it was shown that over‐expression of CXCR4 enhanced the migratory capacity of USSCs. The increase of CXCR4 expression, here caused by VEGF in USSCs, can improve the efficacy of stem cell therapy and transplantation after long‐term culture of stem cells before clinical use.     

Comparative capability of menstrual blood versus bone marrow derived stem cells in neural differentiation

In order to characterize the potency of menstrual blood stem cells (MenSCs) for future cell therapy of neurological disorders instead of bone marrow stem cells (BMSCs) as a well-known and conventional source of adult stem cells, we examined the in vitro differentiation potential of these stem cells into neural-like cells. The differentiation potential of MenSCs to neural cells in comparison with BMSCs was assessed under two step neural differentiation including conversion to neurosphere-like cells and final differentiation. The expression levels of Nestin, Microtubule-associated protein 2, gamma-aminobutyric acid type B receptor subunit 1 and 2, and Tubulin, beta 3 class III mRNA and/or protein were up-regulated during development of MenSCs into neurosphere-like cells (NSCs) and neural-like cells. The up-regulation level of these markers in differentiated neural-like cells from MenSCs was comparable with differentiated cells from BMSCs. Moreover, both differentiated MenSCs and BMSCs expressed high levels of potassium, calcium and sodium channel genes developing functional channels with electrophysiological recording. For the first time, we demonstrated that MenSCs are a unique cell population with differentiation ability into neural-like cells comparable to BMSCs. In addition, we have introduced an approach to generate NSCs from MenSCs and BMSCs and their further differentiation into neural-like cells in vitro. Our results hold a promise to future stem cell therapy of neurological disorders using NSCs derived from menstrual blood, an accessible source in every woman.     

Comparison of long-term retinoic acid-based neural induction methods of bone marrow human mesenchymal stem cells

The differentiation of human mesenchymal stem cells (hMSCs) into neural cells in vitro provides a potential tool to be utilized for cell therapy of neurodegenerative disorders. Although previous studies repeated different protocols for the induction of neural cells from hMSCs in vitro, the results were not in complete agreement. In this study, we have attempted to compare three of these neural induction methods; retinoic acid (RA) treatment, RA treatment in serum reduced conditions, and treatment using other chemical compounds (dimethyl sulfoxide and potassium chloride) along with RA by real-time cell analysis and immunofluorescent staining of neural markers. RA treatment led to a slow progression of cells into neural-like morphology with the expression of neural protein neurofilament whereas reducing serum during RA treatment caused a much more extended differentiation process. Additionally, neural-like morphology was persistent in the later periods of differentiation in RA treatment. On the other hand, chemical induction caused cell shrinkages mimicking neural-like morphology in a short time and loss of this morphology along with increased cell death in later periods. Among the three methods compared, RA treatment was the most reliable one in terms of stability of differentiation and neural protein expressions.     

From spindle to spherical: Is spherical shape a potential predictor of human mesenchymal stem cells with increased differentiation capability?

Because human mesenchymal stem cells (hMSCs) can proliferate indefinitely in an undifferentiated state and differentiate into various cell types, hMSCs are expected to be useful for cell replacement therapy. But the clinic application is limited by its differentiation efficiency of hMSCs. It has been proved that cells can be geometrically switched between gene programs for growth, apoptosis and differentiation. Previous studies showed that hMSCs started showing round when exposed to modeled microgravity (MMG), while their differentiation capability seemed enhanced simultaneously. Thus, this article briefly reviews such studies, and hypothesizes that “spherical shape” could be a potential predictor of hMSCs with potentiated differentiation capability.     

Stabilization of cellular properties and differentiation mutilpotential of human mesenchymal stem cells transduced with hTERT gene in a long-term culture

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.     

Transplantation of neuronal-primed human bone marrow mesenchymal stem cells in hemiparkinsonian rodents

Bone marrow-derived human mesenchymal stem cells (hMSCs) have shown promise in in vitro neuronal differentiation and in cellular therapy for neurodegenerative disorders, including Parkinson' disease. However, the effects of intracerebral transplantation are not well defined, and studies do not agreed on the optimal neuronal differentiation method. Here, we investigated three growth factor-based neuronal differentiation procedures (using FGF-2/EGF/PDGF/SHH/FGF-8/GDNF), and found all to be capable of eliciting an immature neural phenotype, in terms of cell morphology and gene/protein expression. The neuronal-priming (FGF-2/EGF) method induced neurosphere-like formation and the highest NES and NR4A2 expression by hMSCs. Transplantation of undifferentiated and neuronal-primed hMSCs into the striatum and substantia nigra of 6-OHDA-lesioned hemiparkinsonian rats revealed transient graft survival of 7 days, despite the reported immunosuppressive properties of MSCs and cyclosporine-immunosuppression of rats. Neither differentiation of hMSCs nor induction of host neurogenesis was observed at injection sites, and hMSCs continued producing mesodermal fibronectin. Strategies for improving engraftment and differentiation post-transplantation, such as prior in vitro neuronal-priming, nigral and striatal grafting, and co-transplantation of olfactory ensheathing cells that promote neural regeneration, were unable to provide advantages. Innate inflammatory responses (Iba-1-positive microglia/macrophage and GFAP-positive astrocyte activation and accumulation) were detected around grafts within 7 days. Our findings indicate that growth factor-based methods allow hMSC differentiation toward immature neuronal-like cells, and contrary to previous reports, only transient survival and engraftment of hMSCs occurs following transplantation in immunosuppressed hemiparkinsonian rats. In addition, suppression of host innate inflammatory responses may be a key factor for improving hMSC survival and engraftment.     

Liver engraftment potential of hepatic cells derived from patient-specific induced pluripotent stem cells

Human induced pluripotent stem cells (iPSCs) are potential renewable sources of hepatocytes for drug development and cell therapy. Differentiation of human iPSCs into different developmental stages of hepatic cells has been achieved and improved during the last several years. We have recently demonstrated the liver engraftment and regenerative capabilities of human iPSC-derived multistage hepatic cells in vivo. Here we describe the in vitro and in vivo activities of hepatic cells derived from patientspecific iPSCs, including multiple lines established from either inherited or acquired liver diseases, and discuss basic and clinical applications of these cells for disease modeling, drug screening and discovery, gene therapy and cell replacement therapy.Key words: induced pluripotent stem cells (iPSCs), hepatic differentiation, liver ngraftment, disease modeling, drug testing, alpha-1 antitrypsin, liver cirrhosis, hepatocellular carcinoma, cell therapy     

Growth factor-defined culture medium for human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) are potential cellular sources of therapeutic stem cells as they have the ability to proliferate and differentiate into a wide array of mesenchymal cell types such as osteoblasts, chondroblasts and adipocytes. hMSCs have been used clinically to treat patients with graft vs. host disease, osteogenesis imperfect, or alveolar cleft, suggesting that transplantation of hMSCs is comparatively safe as a stem cell-based therapy. However, conventional culture medium for hMSCs contains fetal bovine serum (FBS). In the present study, we developed a growth factor-defined, serum-free medium for culturing hMSCs. Under these conditions, TGF-beta1 promoted proliferation of hMSCs. The expanded hMSC population expressed the human pluripotency markers SSEA-3, -4, NANOG, OCT3/4 and SOX2. Furthermore, double positive cells for SSEA-3 and a mesenchymal cell marker, CD105, were detected in the population. The potential to differentiate into osteoblasts and adipocytes was confirmed. This work provides a useful tool to understand the basic biological properties of hMSCs in culture.     

Development and evaluation of a trehalose-contained solution formula to preserve hUC-MSCs at 4°C

Human mesenchymal stem cells (hMSCs) hold great promise in cell therapy and regenerative medicine. Various preclinical and clinical trials have been carried out to illustrate the therapeutic potential of these cells. However, one major challenge for manufacturing clinical grade hMSCs is the requisition of current good manufacturing practice (cGMP) grade practices in cell isolation, processing, storage, and distribution. Development of non-toxic and animal serum-free preservation medium is critical for storage and distribution of mesenchymal stem cells (MSCs). In this study, we developed a solution formula that could preserve MSCs at 4°C for up to 3 weeks. In the solution, trehalose is a key ingredient for maintaining survival of MSCs. Among the concentrations investigated, 40 mM trehalose showed the best outcome with the viability maintained more than 92.7 ± 1.5% for 7 days. Cells preserved in the solution formula for 3 weeks still remained about 70% viability, and produced results similar to those of freshly harvested hMSCs in terms of growth kinetics, expression profile of cell surface antigens, and differentiation potential. In summary, storage of MSCs in the medium makes it far easier for transporting the cells from processing units to clinical sites.     

Osteostimulation scaffolds of stem cells: BMP‐7‐derived peptide‐decorated alginate porous scaffolds promote the aggregation and osteo‐differentiation of human mesenchymal stem cells

The scaffolds for stem cell‐based bone tissue engineering should hold the ability to guide stem cells osteo‐differentiating. Otherwise, stem cells will differentiate into unwanted cell types or will form tumors in vivo. Alginate, a natural polysaccharide with great biocompatibility, was widely used in biomedical applications. However, the limited bioactivity and poor osteogenesis capability of pristine alginate hampered its further application in tissue engineering. In this work, a bone forming peptide‐1 (BFP‐1), derived from bone morphogenetic protein‐7, was grafted to alginate polymer chains to prepare peptide‐decorated alginate porous scaffolds (pep‐APS) for promoting osteo‐differentiation of human mesenchymal stem cells (hMSCs). SEM images of pep‐APS exhibited porous structure with about 90% porosity (pore size 100–300 μm), which was appropriate for hMSCs ingrowth. The adhesion, proliferation and aggregation of hMSCs grown on pep‐APS were enhanced in vitro. Moreover, pep‐APS promoted the alkaline phosphatase (ALP) activity of hMSCs, and the osteo‐related genes expression was obviously up‐regulated. The immunochemical staining and western blot analysis results showed high expression level of OCN and Col1a1 in the hMSCs grown on pep‐APS. This work provided a facile and valid strategy to endow the alginate polymers themselves with specific bioactivity and prepare osteopromoting scaffold with enhanced osteogenesis ability, possessing potential applications in stem cell therapy and regenerative medicine.     

miR-335 orchestrates cell proliferation, migration and differentiation in human mesenchymal stem cells

In spite of the extensive potential of human mesenchymal stem cells (hMSCs) in cell therapy, little is known about the molecular mechanisms that regulate their therapeutic properties. We aimed to identify microRNAs (miRNAs) involved in controlling the transition between the resting and reparative phenotypes of hMSCs, hypothesizing that these miRNAs must be present in the undifferentiated cells and downregulated to allow initiation of distinct activation/differentiation programs. Differential miRNA expression analyses revealed that miR-335 is significantly downregulated upon hMSC differentiation. In addition, hMSCs derived from a variety of tissues express miR-335 at a higher level than human skin fibroblasts, and overexpression of miR-335 in hMSCs inhibited their proliferation and migration, as well as their osteogenic and adipogenic potential. Expression of miR-335 in hMSCs was upregulated by the canonical Wnt signaling pathway, a positive regulator of MSC self-renewal, and downregulated by interferon-γ (IFN-γ), a pro-inflammatory cytokine that has an important role in activating the immunomodulatory properties of hMSCs. Differential gene expression analyses, in combination with computational searches, defined a cluster of 62 putative target genes for miR-335 in hMSCs. Western blot and 3'UTR reporter assays confirmed RUNX2 as a direct target of miR-335 in hMSCs. These results strongly suggest that miR-335 downregulation is critical for the acquisition of reparative MSC phenotypes.     

The Path from Skin to Brain: Generation of Functional Neurons from Fibroblasts

Cell fate reprogramming makes possible the generation of new cell types from healthy adult cells to replace those lost or damaged in disease. Additionally, reprogramming patient cells into specific cell types allows for drug screening and the development of new therapeutic tools. Generation of new neurons is of particular interest because of the potential to treat diseases of the nervous system, such as neurodegenerative disorders and spinal cord injuries, with cell replacement therapy. Recent advances in cell fate reprogramming have led to the development of novel methods for the direct conversion of fibroblasts into neurons and neural stem cells. This review will highlight the advantages of these new methods over neuronal induction from embryonic stem cells and induced pluripotent stem cells, as well as outline the limitations and the potential for future applications.     

Pluripotent Stem Cells Models for Huntington＇s Disease： Prospects and Challenges

Pluripotent cellular models have shown great promise in the study of a number of neurological disorders.Several advantages of using a stem cell model include the potential for cells to derive disease relevant neuronal cell types,providing a system for researchers to monitor disease progression during neurogenesis,along with serving as a platform for drug discovery.A number of stem cell derived models have been employed to establish in vitro research models of Huntington’s disease that can be used to investigate cellular pathology and screen for drug and cell-based therapies.Although some progress has been made,there are a number of challenges and limitations that must be overcome before the true potential of this research strategy is achieved.In this article we review current stem cell models that have been reported,as well as discuss the issues that impair these studies.We also highlight the prospective application of Huntington’s disease stem cell models in the development of novel therapeutic strategies and advancement of personalized medicine.