Identification of Rat Respiratory Mucosa Stem Cells and Comparison of the Early Neural Differentiation Potential with the Bone Marrow Mesenchymal Stem Cells In Vitro

The aim of this study is to identify rat nasal septum respiratory mucosa-derived mesenchyme stem cells (RM-MSCs) and to compare its neural lineage differentiation capacity with bone marrow-derived mesenchyme stem cells (BM-MSCs) after a short period of neural induction culture in vitro. The cell morphology was observed with light microscopy; cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The characteristics of the cells were evaluated with flow cytometry, immunofluorescence, real-time quantitative PCR (RT-PCR), and Western blotting. The results showed that rat nasal respiratory mucosa contains RM-MSCs that exhibited similar proliferation rate as BM-MSCs in vitro. Both RT-PCR and Western blotting analyses demonstrated that RM-MSCs showed higher expression of neural lineage markers than BM-MSCs after a short period of neural induction culture, and secreted higher level of brain-derived neurotrophic factor. RM-MSCs were more amenable to differentiate into neural or glial cell after a short period of neural induction culture than BM-MSCs in vitro; and it could be considered as another optimal source of stem cells for cell-based therapy to neurological diseases.     

Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells

Multipotent and self-renewing neural stem cells have been isolated in culture, but equivalent cells have not yet been prospectively identified in neural tissue. Using cell surface markers and flow cytometry, we have isolated neural crest stem cells (NCSCs) from mammalian fetal peripheral nerve. These cells are phenotypically and functionally indistinguishable from NCSCs previously isolated by culturing embryonic neural tube explants. Moreover, in vivo BrdU labeling indicates that these stem cells self-renew in vivo. NCSCs freshly isolated from nerve tissue can be directly transplanted in vivo, where they generate both neurons and glia. These data indicate that neural stem cells persist in peripheral nerve into late gestation by undergoing self-renewal. Such persistence may explain the origins of some PNS tumors in humans.     

Neural crest‐derived stem cells display a wide variety of characteristics

A recent burst of findings has shown that neural crest‐derived stem cells (NCSCs) can be found in diverse mammalian tissues. In addition to their identification in tissues that are known to be derived from the neural crest, recent studies have revealed NCSCs in tissues that are not specifically derived from the neural crest, such as bone marrow. NCSCs can express a wide range of characteristics, and which properties are expressed mainly depends on their tissue sources and the ontogenic stage of the animal. The identification of NCSCs in various tissues opens an entirely new avenue of approach to developing autologous cell replacement therapies for use in regenerative medicine. In this review, we discuss the origin, migration, and lineage potential of NCSCs from various mammalian tissue sources. J. Cell. Biochem. 107: 1046–1052, 2009. © 2009 Wiley‐Liss, Inc.     

Ontogeny and multipotency of neural crest-derived stem cells in mouse bone marrow, dorsal root ganglia, and whisker pad

Although recent reports have described multipotent, self-renewing, neural crest-derived stem cells (NCSCs), the NCSCs in various adult rodent tissues have not been well characterized or compared. Here we identified NCSCs in the bone marrow (BM), dorsal root ganglia, and whisker pad and prospectively isolated them from adult transgenic mice encoding neural crest-specific P0-Cre/Floxed-EGFP and Wnt1-Cre/Floxed-EGFP. Cultured EGFP-positive cells formed neurosphere-like structures that expressed NCSC genes and could differentiate into neurons, glial cells, and myofibroblasts, but the frequency of the cell types was tissue source dependent. Interestingly, we observed NCSCs in the aorta-gonad-mesonephros region, circulating blood, and liver at the embryonic stage, suggesting that NCSCs migrate through the bloodstream to the BM and providing an explanation for how neural cells are generated from the BM. The identification of NCSCs in accessible adult tissue provides a new potential source for autologous cell therapy after nerve injury or disease.     

Intrinsic differences among spatially distinct neural crest stem cells in terms of migratory properties, fate determination, and ability to colonize the enteric nervous system

We have systematically examined the developmental potential of neural crest stem cells from the enteric nervous system (gut NCSCs) in vivo to evaluate their potential use in cellular therapy for Hirschsprung disease and to assess differences in the properties of postmigratory NCSCs from different regions of the developing peripheral nervous system (PNS). When transplanted into developing chicks, flow-cytometrically purified gut NCSCs and sciatic nerve NCSCs exhibited intrinsic differences in migratory potential and neurogenic capacity throughout the developing PNS. Most strikingly, gut NCSCs migrated into the developing gut and formed enteric neurons, while sciatic nerve NCSCs failed to migrate into the gut or to make enteric neurons, even when transplanted into the gut wall. Enteric potential is therefore not a general property of NCSCs. Gut NCSCs also formed cholinergic neurons in parasympathetic ganglia, but rarely formed noradrenergic sympathetic neurons or sensory neurons. Supporting the potential for autologous transplants in Hirschsprung disease, we observed that Endothelin receptor B (Ednrb)-deficient gut NCSCs engrafted and formed neurons as efficiently in the Ednrb-deficient hindgut as did wild-type NCSCs. These results demonstrate intrinsic differences in the migratory properties and developmental potentials of regionally distinct NCSCs, indicating that it is critical to match the physiological properties of neural stem cells to the goals of proposed cell therapies.     

Effects of low-intensity pulsed ultrasound on cell viability, proliferation and neural differentiation of induced pluripotent stem cells-derived neural crest stem cells

Low-intensity pulsed ultrasound (LIPUS) acting on induced pluripotent stem cells–derived neural crest stem cells (iPSCs–NCSCs) is considered a promising therapy to improve the efficacy of injured peripheral nerve regeneration. Effects of LIPUS on cell viability, proliferation and neural differentiation of iPSCs–NCSCs were examined respectively in this study. LIPUS at 500 mW cm^?2 enhanced the viability and proliferation of iPSCs–NCSCs after 2 days and, after 4 days, up-regulated gene and protein expressions of NF-M, Tuj1, S100β and GFAP in iPSCs–NCSCs whereas after 7 days expression of only NF-M, S100β and GFAP were up-regulated. LIPUS treatment at an appropriate intensity can, therefore, be an efficient and cost-effective method to enhance cell viability, proliferation and neural differentiation of iPSCs–NCSCs in vitro for peripheral nerve tissue engineering.     

Matrix stiffness modulates the differentiation of neural crest stem cells in vivo

Stem cells are often transplanted with scaffolds for tissue regeneration; however, how the mechanical property of a scaffold modulates stem cell fate in vivo is not well understood. Here we investigated how matrix stiffness modulates stem cell differentiation in a model of vascular graft transplantation. Multipotent neural crest stem cells (NCSCs) were differentiated from induced pluripotent stem cells, embedded in the hydrogel on the outer surface of nanofibrous polymer grafts, and implanted into rat carotid arteries by anastomosis. After 3 months, NCSCs differentiated into smooth muscle cells (SMCs) near the outer surface of the polymer grafts; in contrast, NCSCs differentiated into glial cells in the most part of the hydrogel. Atomic force microscopy demonstrated a stiffer matrix near the polymer surface but much lower stiffness away from the polymer graft. Consistently, in vitro studies confirmed that stiff surface induced SMC genes whereas soft surface induced glial genes. These results suggest that the scaffold’s mechanical properties play an important role in directing stem cell differentiation in vivo, which has important implications in biomaterials design for stem cell delivery and tissue engineering.     

Adult mesenchymal stem cells and cell-based tissue engineering

The identification of multipotential mesenchymal stem cells (MSCs) derived from adult human tissues, including bone marrow stroma and a number of connective tissues, has provided exciting prospects for cell-based tissue engineering and regeneration. This review focuses on the biology of MSCs, including their differentiation potentials in vitro and in vivo, and the application of MSCs in tissue engineering. Our current understanding of MSCs lags behind that of other stem cell types, such as hematopoietic stem cells. Future research should aim to define the cellular and molecular fingerprints of MSCs and elucidate their endogenous role(s) in normal and abnormal tissue functions.     

Adult mesenchymal stem cells and cell-based tissue engineering

The identification of multipotential mesenchymal stem cells (MSCs) derived from adult human tissues, including bone marrow stroma and a number of connective tissues, has provided exciting prospects for cell-based tissue engineering and regeneration. This review focuses on the biology of MSCs, including their differentiation potentials in vitro and in vivo, and the application of MSCs in tissue engineering. Our current understanding of MSCs lags behind that of other stem cell types, such as hematopoietic stem cells. Future research should aim to define the cellular and molecular fingerprints of MSCs and elucidate their endogenous role(s) in normal and abnormal tissue functions.     

Neural crest stem cells persist in the adult gut but undergo changes in self-renewal,neuronal subtype potential,and factor responsiveness

We found neural crest stem cells (NCSCs) in the adult gut. Postnatal gut NCSCs were isolated by flow-cytometry and compared to fetal gut NCSCs. They self-renewed extensively in culture but less than fetal gut NCSCs. Postnatal gut NCSCs made neurons that expressed a variety of neurotransmitters but lost the ability to make certain subtypes of neurons that are generated during fetal development. Postnatal gut NCSCs also differed in their responsiveness to lineage determination factors, affecting cell fate determination in vivo and possibly explaining their reduced neuronal subtype potential. These perinatal changes in gut NCSCs parallel perinatal changes in hematopoietic stem cells, suggesting that stem cells in different tissues undergo similar developmental transitions. The persistence of NCSCs in the adult PNS opens up new possibilities for regeneration after injury or disease.     

The role of recipient T cells in mesenchymal stem cell-based tissue regeneration

Significant progress has been made in stem cell biology, regenerative medicine, and stem cell-based tissue engineering. Such scientific strides highlight the potential of replacing or repairing damaged tissues in congenital abnormalities, diseases, or injuries, as well as constructing functional tissue or organs in vivo. Since mesenchymal stem cells (MSCs) are capable of differentiating into bone-forming cells, they constitute an appropriate cell source to repair damaged bone tissues. In addition, the immunoregulatory property of MSCs provides a foundation for their use in treating a variety of autoimmune diseases. However, the interaction between MSCs and immune cells in cell-based tissue regeneration is largely unknown. In this review, we will discuss the current understanding of MSC-based tissue regeneration, emphasizing the role of the immune microenvironment in bone regeneration.     

Induction of multipotency in umbilical cord-derived mesenchymal stem cells cultivated under suspension conditions

Due to the limitations in the clinical application of embryonic stem cells (ESC) and induced pluripotent stem cells, mesenchymal stem cells (MSCs) are now much more interesting for cell-based therapy. Although MSCs have several advantages, they are not capable of differentiating to all three embryonic layers (three germ layers) without cultivation under specific induction media. Hence, improvement of MSCs for cell therapy purposes is under intensive study now. In this study, we isolated MSCs from umbilical cord tissue at the single-cell level, by treatment with trypsin, followed by cultivation under suspension conditions to form a colony. These colonies were trypsin resistant, capable of self-renewal differentiation to the three germ layers without any induction, and they were somewhat similar to ESC colonies. The cells were able to grow in both adherent and suspension culture conditions, expressed both the MSCs markers, especially CD105, and the multipotency markers, i.e., SSEA-3, and had a limited lifespan. The cells were expanded under simple culture conditions at the single-cell level and were homogenous. Further and complementary studies are required to understand how trypsin-tolerant mesenchymal stem cells are established. However, our study suggested non-embryonic resources for future cell-based therapy.     

Cell-intrinsic differences between stem cells from different regions of the peripheral nervous system regulate the generation of neural diversity

Stem cells in different regions of the nervous system give rise to different types of mature cells. This diversity is assumed to arise in response to local environmental differences, but the contribution of cell-intrinsic differences between stem cells has been unclear. At embryonic day (E)14, neural crest stem cells (NCSCs) undergo primarily neurogenesis in the gut but gliogenesis in nerves. Yet gliogenic and neurogenic factors are expressed in both locations. NCSCs isolated by flow-cytometry from E14 sciatic nerve and gut exhibited heritable, cell-intrinsic differences in their responsiveness to lineage determination factors. Gut NCSCs were more responsive to neurogenic factors, while sciatic nerve NCSCs were more responsive to gliogenic factors. Upon transplantation of uncultured NCSCs into developing peripheral nerves in vivo, sciatic nerve NCSCs gave rise only to glia, while gut NCSCs gave rise primarily to neurons. Thus, cell fate in the nerve was stem cell determined.     

Impact of Aging on the Regenerative Properties of Bone Marrow-, Muscle-, and Adipose-Derived Mesenchymal Stem/Stromal Cells

Mesenchymal stem/stromal cells (MSCs) are promising cell sources for regenerative therapies due to their multipotency and ready availability, but their application can be complicated by patient-specific factors like age or illness. MSCs have been investigated for the treatment of many musculoskeletal disorders, including osteoarthritis and osteoporosis. Due to the prevalence of these diseases in older populations, researchers have studied how aging affects MSC properties and have found that proliferation and differentiation potential are impaired. However, these effects have never been compared among MSCs isolated from multiple tissue sources in the same, healthy donor. Revealing differences in how MSCs are affected by age could help identify an optimal cell source for musculoskeletal therapies targeting older patients. MSCs were isolated from young and old rabbit bone marrow, muscle, and adipose tissue. Cell yield and viability were quantified after isolation procedures, and expansion properties were assessed using assays for proliferation, senescence, and colony formation. Multipotency was also examined using lineage-specific stains and spectrophotometry of metabolites. Results were compared between age groups and among MSC sources. Results showed that MSCs are differentially influenced by aging, with bone marrow-derived stem cells having impaired proliferation, senescence, and chondrogenic response, whereas muscle-derived stem cells and adipose-derived stem cells exhibited no negative effects. While age reduced overall cell yield and adipogenic potential of all MSC populations, osteogenesis and clonogenicity remained unchanged. These findings indicate the importance of age as a factor when designing cell-based therapies for older patients.     

Immortalized mesenchymal stem cells: an alternative to primary mesenchymal stem cells in neuronal differentiation and neuroregeneration associated studies

Background  

Mesenchymal stem cells (MSCs) can be induced to differentiate into neuronal cells under appropriate cellular conditions and transplanted in brain injury and neurodegenerative diseases animal models for neuroregeneration studies. In contrast to the embryonic stem cells (ESCs), MSCs are easily subject to aging and senescence because of their finite ability of self-renewal. MSCs senescence seriously affected theirs application prospects as a promising tool for cell-based regenerative medicine and tissue engineering. In the present study, we established a reversible immortalized mesenchymal stem cells (IMSCs) line by using SSR#69 retrovirus expressing simian virus 40 large T (SV40T) antigen as an alternative to primary MSCs.     

Uniaxial mechanical strain modulates the differentiation of neural crest stem cells into smooth muscle lineage on micropatterned surfaces

Neural crest stem cells (NCSCs) play an important role in the development and represent a valuable cell source for tissue engineering. However, how mechanical factors in vivo regulate NCSC differentiation is not understood. Here NCSCs were derived from induced pluripotent stem cells and used as a model to determine whether vascular mechanical strain modulates the differentiation of NCSCs into smooth muscle (SM) lineage. NCSCs were cultured on micropatterned membranes to mimic the organization of smooth muscle cells (SMCs), and subjected to cyclic uniaxial strain. Mechanical strain enhanced NCSC proliferation and ERK2 phosphorylation. In addition, mechanical strain induced contractile marker calponin-1 within 2 days and slightly induced SM myosin within 5 days. On the other hand, mechanical strain suppressed the differentiation of NCSCs into Schwann cells. The induction of calponin-1 by mechanical strain was inhibited by neural induction medium but further enhanced by TGF-β. For NCSCs pre-treated with TGF-β, mechanical strain induced the gene expression of both calponin-1 and SM myosin. Our results demonstrated that mechanical strain regulates the differentiation of NCSCs in a manner dependent on biochemical factors and the differentiation stage of NCSCs. Understanding the mechanical regulation of NCSC differentiation will shed light on the development and remodeling of vascular tissues, and how transplanted NCSCs respond to mechanical factors.     

脐静脉和骨髓来源的间充质干细胞的比较研究

间充质干细胞(MSCs)的来源有限,成人骨髓是MSCs的主要来源,这极大地限制了其在实验和临床中的应用。为拓宽MSCs来源,从细胞形态、生长特性、免疫表型和多向分化能力等四个方面对人脐静脉来源和成人骨髓来源的间充质干细胞进行了比较研究。结果表明,人脐静脉来源和成人骨髓来源的 MSCs具有相似的生物学特征,成纤维细胞样形态生长,并具有强大的体外扩增和多向分化能力。人脐静脉来源的MSCs可替代成人骨髓MSCs,作为满足实验和临床需要的重要来源。     

The roles of mesenchymal stem cells (MSCs) therapy in ischemic heart diseases

Growing cell-based myocardial therapies which could lead to successful myocardial repair attracts medical interest. Even more intriguing is the observation that MSCs appears to be a more potent material among kinds of stem cells for the transplantation, the mechanism for this benefit remains unclear. However, the therapeutic contribution of MSCs to myocardial repair can be caused by multiple factors including: direct differentiation into cardiac tissue including cardiomyocytes, smooth muscle cell, and vascular endothelial cells; secreting a variety of cytokines and growth factors that have paracrine activities; spontaneous cell fusion; and stimulating endogenous repair. In addition, MSCs possess local immunosuppressive properties, and MSCs mobilization is widely used clinically for transplantation. We will discusses the potential mechanisms of MSCs repair for ischemic heart diseases.     

Fine‐tuning of dual‐SMAD inhibition to differentiate human pluripotent stem cells into neural crest stem cells

ObjectivesThe derivation of neural crest stem cells (NCSCs) from human pluripotent stem cells (hPSCs) has been commonly induced by WNT activation in combination with dual‐SMAD inhibition.In this study, by fine‐tuning BMP signalling in the conventional dual‐SMAD inhibition, we sought to generate large numbers of NCSCs without WNT activation.Materials and methodsIn the absence of WNT activation, we modulated the level of BMP signalling in the dual‐SMAD inhibition system to identify conditions that efficiently drove the differentiation of hPSCs into NCSCs. We isolated two NCSC populations separately and characterized them in terms of global gene expression profiles and differentiation ability.ResultsOur modified dual‐SMAD inhibition containing a lower dose of BMP inhibitor than that of the conventional dual‐SMAD inhibition drove hPSCs into mainly NCSCs, which consisted of HNK⁺p75high and HNK⁺p75low cell populations. We showed that the p75high population formed spherical cell clumps, while the p75low cell population generated a 2D monolayer.We detected substantial differences in gene expression profiles between the two cell groups and showed that both p75high and p75low cells differentiated into mesenchymal stem cells (MSCs), while only p75high cells had the ability to become peripheral neurons.ConclusionsThis study will provide a framework for the generation and isolation of NCSC populations for effective cell therapy for peripheral neuropathies and MSC‐based cell therapy.