The evaluation of cyclic uniaxial strain on myogenic differentiation of adipose-derived stem cells

It has been revealed that skeletal muscle cells have the potential to generate, sense and respond to biomechanical signals and that, mechanical force is one of the important factors influencing proliferation, differentiation, regeneration and homeostasis of skeletal muscle cells and myoblasts. The aim of this study was to illustrate the effect of cyclic uniaxial strain on myogenic differentiation of adipose-derived stem cells (ASCs). This study was designed to investigate this effect within 3 days in 4 groups: control (untreated), chemical, chemical-mechanical and mechanical based on exposure of ASCs to chemical growth factors for 3 days or to mechanical strain just on the 2nd day. Finally, cell orientation, muscle-related gene expression, myosin protein synthesis and the number of myosin-positive cells were examined to estimate the rate of differentiation. By studying the cells before and after exposure to uniaxial strain, it could be observed that by exerting the load, the cells were organized almost perpendicularly to strain direction. Real-time RT-PCR demonstrated that uniaxial strain had a significant effect on up-regulation of muscle-related genes in chemical–mechanical group (P < 0.001) as compared to mechanical or chemical groups. Immunocytochemistry confirmed the myosin-positive cells in treated groups and the numbers of these cells were enumerated by flow cytometry. These data suggest that uniaxial cyclic strain could affect ASCs and cause their myogenic differentiation and that the combination of chemical myogenic differentiation factors with mechanical signals promotes differentiation much more than differentiation by chemical myogenic differentiation factors or mechanical signals alone.     

PKC-delta induces cardiomyogenic gene expression in human adipose-derived stem cells

Stromal cells from fat tissues exhibit properties of mesenchymal stem cells from other sources with the ability to differentiate towards multiple cell types. However, effective differentiation of these mesenchymal cells, called adipose-derived stem cells (ADSCs), towards cardiomyogenic lineage has been limited to a small number of isolated clones in an extended culture. Previously, we reported that treatment with phorbol ester induces the expression of several cardiomyogenic genes in the absence of serum. This study was performed to identify the roles of PKC isoforms in cardiomyogenic gene expression of ADSCs. Treatment with 10 nM phorbol myristate acetate (PMA) for 24 h caused sustained increases in mRNA levels for various cardiomyogenic genes, such as Mef2C, cardiac actin and troponin, for at least 6 days following the drug removal. The use of various inhibitors specific for PKC isoforms demonstrated that the novel PKC-theta/delta isoforms mediate the PMA effects. RT-PCR revealed that ADSCs express significant mRNA for PKC-delta, but not theta isoform. Overexpression of cDNA for PKC-delta resulted in marked increases in cardiac mRNA expression. These results indicate that activation of PKC-delta induces the expression of multiple cardiomyogenic genes in ADSCs.     

Comparing the Effect of Uniaxial Cyclic Mechanical Stimulation and Chemical Factors on Myogenin and Myh2 Expression in Mouse Embryonic and Bone Marrow Derived Mesenchymal Stem Cells

Background: Environmental factors affect stem cell differentiation. In addition to chemical factors, mechanical signals have been suggested to enhance myogenic differentiation of stem cells. Therefore, this study was undertaken to illustrate and compare the effect of chemical and mechanical stimuli on Myogenin (MyoG) and Myosin heavy chani 2 (Myh2) expression of mouse bone marrowderived mesenchymal stem cells (BMSCs) and embryonic stem cells (ESCs). Methods: After isolation and expansion of BMSCs and generation of embryoid bodies and spontaneous differentiation of ESCs, cells were examined in 4 groups: (1) control group: untreated cells; (2) chemical group: cells incubated in myogenic medium (5-azacythidine and horse serum for BMSCs, dimethyl sulfoxide (DMSO) and horse serum for ESCs) for 5 days; (3) mechanical group: cells exposed to uniaxial cyclic strain (8%, 1 Hz, 24 h) and (4) chemical + mechanical group: cells incubated in myogenic medium for 4 days and then exposed to uniaxial cyclic strain. Real-time PCR was used to examine the expression of MyoG and Myh2 as specific myogenic markers. Results: suggested that mechanical loading, as a single factor, could elevate MyoG and Myh2 expression. Combining chemical with mechanical factor increases expression and there was no significant difference in MyoG expression of ESCs- and MSCs-chemical + mechanical groups; however, Myh2 expression was significantly higher in ESCs-mechanical group than that in the same group of MSCs.     

Growth and osteogenic differentiation of adipose-derived and bone marrow-derived stem cells on chitosan and chitooligosaccharide films

Very low molecular weight chitooligosaccharide (COS, 1.4 kDa) and high molecular weight chitosan (1000 kDa) were comparatively studied in terms of physical and biological characteristics. Thin films of COS, chitosan and gelatin were prepared and crosslinked by dehydrothermal treatment at 140 °C for 24 h. COS film presented more hydrophilic property than chitosan film. Behaviors of rat adipose-derived stem cells (ASCs) and bone marrow-derived stem cells (MSCs) were investigated on COS and chitosan films, comparing to those on gelatin film. The results on cell spreading suggested that both ASCs and MSCs preferred to attach on COS film than chitosan film with 6–7 times larger cell areas. Numbers of both stem cells proliferated on COS film were approximately 3-fold higher than those on chitosan film. In addition, COS film enhanced osteogenic differentiating potential of MSCs, as observed from the alkaline phosphatase activity and calcium deposition. Therefore, in this work, COS was shown to be a more favorable material for the growth and osteogenic differentiation of both ASCs and MSCs, compared to high molecular weight chitosan.     

Constitutive overexpression of the integral membrane protein Itm2A enhances myogenic differentiation of C2C12 cells

Integral membrane protein 2A (Itm2A) is a transmembrane protein belonging to a family composed of at least two other members, Itm2B and Itm2C, all of them having a different expression pattern. The protein serves as a marker for early stages in chondrogenesis and T-cell development. Itm2A is also highly expressed in skeletal muscle. In order to understand the role of Itm2A in muscle development, we constitutively overexpressed exogenous Itm2A in C2C12 myoblast cells. Several clones expressing high levels of Itm2a were isolated and characterized. Overexpression was associated with enhanced tube formation and the appearance of multinuclear cells. Gene expression analysis demonstrated that muscle creatin kinase was upregulated in the presence of exogenous Itm2A. Interestingly, proliferation rates were not altered in the undifferentiated myoblast C2C12 cells. These results demonstrate that overexpression of Itm2a in C2C12 enhances myogenic differentiation in vitro.     

Signals from damaged but not undamaged skeletal muscle induce myogenic differentiation of rat bone-marrow-derived mesenchymal stem cells

The regenerative capacity of skeletal muscle has been usually attributed to resident satellite cells, which, upon activation by local or distant stimuli, initiate a myogenic differentiation program. Although recent studies have revealed that bone-marrow-derived progenitor cells may also participate in regenerative myogenesis, the signals and mechanisms involved in this process have not been elucidated. This study was designed to investigate whether signals from injured rat skeletal muscle were competent to induce a program of myogenic differentiation in expanded cultures of rat bone-marrow-derived mesenchymal stem cells (MSC). We observed that the incubation of MSC with a conditioned medium prepared from chemically damaged but not undamaged muscle resulted in a time-dependent change from fibroblast-like into elongated multinucleated cells, a transient increase in the number of MyoD positive cells, and the subsequent onset of myogenin, alpha-actinin, and myosin heavy chain expression. These results show that damaged rat skeletal muscle is endowed with the capacity to induce myogenic differentiation of bone-marrow-derived mesenchymal progenitors.     

Monolayer cell expansion conditions affect the chondrogenic potential of adipose-derived stem cells

Adipose-derived stem cells (ASCs) are an abundant, readily available population of multipotent progenitor cells that reside in adipose tissue. Isolated ASCs are typically expanded in monolayer on standard tissue culture plastic with a basal medium containing 10% fetal bovine serum. However, recent data suggest that altering the monolayer expansion conditions by using suspension culture plastic, adding growth factors to the medium, or adjusting the seeding density may affect the self-renewal rate, multipotency, and lineage-specific differentiation potential of the ASCs. We hypothesized that variation in any of these expansion conditions would influence the chondrogenic potential of ASCs. ASCs were isolated from human liposuction waste tissue and expanded through two passages with different tissue culture plastic, feed medium, and cell seeding densities. Once expanded, the cells were cast in an agarose gel and subjected to identical chondrogenic culture conditions for 7 days, at which point cell viability, radiolabel incorporation, and gene expression were measured. High rates of matrix synthesis upon chondrogenic induction were mostly associated with smaller cells, as indicated by cell width and area on tissue culture plastic, and it appears that expansion in a growth factor supplemented medium is important in maintaining this morphology. All end-point measures were highly dependent on the specific monolayer culture conditions. These results support the hypothesis that monolayer culture conditions may "prime" the cells or predispose them towards a specific phenotype and thus underscore the importance of early culture conditions in determining the growth and differentiation potential of ASCs.     

骨髓间充质干细胞成肌分化在骨骼肌再生中的应用

骨骼肌良好的再生能力是由于肌卫星细胞的存在,然而肌卫星细胞的数量仅占骨骼肌细胞数量的1%~ 5%,当肌肉损伤时,仅依靠这些卫星细胞还不足以促进骨骼肌修复与再生,并且这种再生能力会随着年龄的增大而衰减,并不能修复损伤严重的骨骼肌。骨髓间充质干细胞（BMSC）因其多向分化潜能,旁分泌潜能,免疫调节能力及容易获取等特点广泛用于损伤骨骼肌的修复与再生。但在某种程度上,仅仅采用BMSC治疗损伤的骨骼肌仍不能达到满意的效果。因此,大量研究采用药物、生物材料、细胞及细胞因子对BMSC进行预处理不仅可改善它的移植率,还可显著促进其向骨骼肌分化,从而最大限度的发掘骨骼肌间充质干细胞的成肌分化潜能以促进骨骼肌的修复。因此,本篇综述旨在概括BMSC成肌分化在骨骼肌再生中的应用。     

Antioxidants cause rapid expansion of human adipose-derived mesenchymal stem cells via CDK and CDK inhibitor regulation

Background

Antioxidants have been shown to enhance the proliferation of adipose-derived mesenchymal stem cells (ADMSCs) in vitro, although the detailed mechanism(s) and potential side effects are not fully understood.In this study, human ADMSCs cultured in ImF-A medium supplemented with antioxidants (N-acetyl-l-cysteine and ascorbic acid-2-phosphate) and fibroblast growth factor 2 (FGF-2) were compared with ADMSCs cultured with FGF-2 alone (ImF) or with FGF-2 under 5% pO₂ conditions (ImF-H).

Results

During log-phase growth, exposure to ImF-A resulted in a higher percentage of ADMSCs in the S phase of the cell cycle and a smaller percentage in G0/G1 phase. This resulted in a significantly reduced cell-doubling time and increased number of cells in the antioxidant-supplemented cultures compared with those supplemented with FGF-2 alone, an approximately 225% higher cell density after 7 days. Western blotting showed that the levels of the CDK inhibitors p21 and p27 decreased after ImF-A treatment, whereas CDK2, CDK4, and CDC2 levels clearly increased. In addition, ImF-A resulted in significant reduction in the expression of CD29, CD90, and CD105, whereas relative telomere length, osteogenesis, adipogenesis, and chondrogenesis were enhanced. The results were similar for ADMSCs treated with antioxidants and those under hypoxic conditions.

Conclusion

Antioxidant treatment promotes entry of ADMSCs into the S phase by suppressing cyclin-dependent kinase inhibitors and results in rapid cell proliferation similar to that observed under hypoxic conditions.     

ARPE-19 conditioned medium promotes neural differentiation of adipose-derived mesenchymal stem cells

BACKGROUNDAdipose-derived stem cells (ASCs) have been increasingly explored for cell-based medicine because of their numerous advantages in terms of easy availability, high proliferation rate, multipotent differentiation ability and low immunogenicity. In this respect, they have been widely investigated in the last two decades to develop therapeutic strategies for a variety of human pathologies including eye disease. In ocular diseases involving the retina, various cell types may be affected, such as Müller cells, astrocytes, photoreceptors and retinal pigment epithelium (RPE), which plays a fundamental role in the homeostasis of retinal tissue, by secreting a variety of growth factors that support retinal cells.AIMTo test ASC neural differentiation using conditioned medium (CM) from an RPE cell line (ARPE-19).METHODSASCs were isolated from adipose tissue, harvested from the subcutaneous region of healthy donors undergoing liposuction procedures. Four ASC culture conditions were investigated: ASCs cultured in basal Dulbecco''s Modified Eagle Medium (DMEM); ASCs cultured in serum-free DMEM; ASCs cultured in serum-free DMEM/F12; and ASCs cultured in a CM from ARPE-19, a spontaneously arising cell line with a normal karyotype derived from a human RPE. Cell proliferation rate and viability were assessed by crystal violet and MTT assays at 1, 4 and 8 d of culture. At the same time points, ASC neural differentiation was evaluated by immunocytochemistry and western blot analysis for typical neuronal and glial markers: Nestin, neuronal specific enolase (NSE), protein gene product (PGP) 9.5, and glial fibrillary acidic protein (GFAP).RESULTSDepending on the culture medium, ASC proliferation rate and viability showed some significant differences. Overall, less dense populations were observed in serum-free cultures, except for ASCs cultured in ARPE-19 serum-free CM. Moreover, a different cell morphology was seen in these cultures after 8 d of treatment, with more elongated cells, often showing cytoplasmic ramifications. Immunofluorescence results and western blot analysis were indicative of ASC neural differentiation. In fact, basal levels of neural markers detected under control conditions significantly increased when cells were cultured in ARPE-19 CM. Specifically, neural marker overexpression was more marked at 8 d. The most evident increase was observed for NSE and GFAP, a modest increase was observed for nestin, and less relevant changes were observed for PGP9.5. CONCLUSIONThe presence of growth factors produced by ARPE-19 cells in tissue culture induces ASCs to express neural differentiation markers typical of the neuronal and glial cells of the retina.     

Reprogramming mediated by stem cell fusion

Advances in mammalian cloning prove that somatic nuclei can be reprogrammed to a state of totipotency by transfer into oocytes. An alternative approach to reprogram the somatic genome involves the creation of hybrids between somatic cells and other cells that contain reprogramming activities. Potential fusion partners with reprogramming activities include embryonic stem cells, embryonic germ cells, embryonal carcinoma cells, and even differentiated cells. Recent advances in fusion-mediated reprogramming are discussed from the standpoints of the developmental potency of hybrid cells, genetic and epigenetic correlates of reprogramming, and other aspects involved in the reprogramming process. In addition, the utility of fusion-mediated reprogramming for future cell-based therapies is discussed.     

Cytokines in adipose-derived mesenchymal stem cells promote the healing of liver disease

Adipose-derived mesenchymal stem cells(ADSCs) are a treatment cell source for patients with chronic liver injury. ADSCs are characterized by being harvested from the patient's own subcutaneous adipose tissue, a high cell yield(i.e., reduced immune rejection response), accumulation at a disease nidus, suppression of excessive immune response, production of various growth factors and cytokines, angiogenic effects, antiapoptotic effects, and control of immune cells via cellcell interaction. We previously showed that conditioned medium of ADSCs promoted hepatocyte proliferation and improved the liver function in a mouse model of acute liver failure. Furthermore, as found by many other groups, the administration of ADSCs improved liver tissue fibrosis in a mouse model of liver cirrhosis. A comprehensive protein expression analysis by liquid chromatography with tandem mass spectrometry showed that the various cytokines and chemokines produced by ADSCs promote the healing of liver disease. In this review, we examine the ability of expressed protein components of ADSCs to promote healing in cell therapy for liver disease. Previous studies demonstrated that ADSCs are a treatment cell source for patients with chronic liver injury. This review describes the various cytokines and chemokines produced by ADSCs that promote the healing of liver disease.     

Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells

BackgroundCell fusion is a phenomenon that is observed in various tissues in vivo, resulting in acquisition of physiological functions such as liver regeneration. Fused cells such as hybridomas have also been produced artificially in vitro. Furthermore, it has been reported that cellular reprogramming can be induced by cell fusion with stem cells.MethodsFused cells between mammalian fibroblasts and mouse embryonic stem cells were produced by electrofusion methods. The phenotypes of each cell lines were analyzed after purifying the fused cells.ResultsColonies which are morphologically similar to mouse embryonic stem cells were observed in fused cells of rabbit, bovine, and zebra fibroblasts. RT-PCR analysis revealed that specific pluripotent marker genes that were never expressed in each mammalian fibroblast were strongly induced in the fused cells, which indicated that fusion with mouse embryonic stem cells can trigger reprogramming and acquisition of pluripotency in various mammalian somatic cells.ConclusionsOur results can help elucidate the mechanism of pluripotency maintenance and the establishment of highly reprogrammed pluripotent stem cells in various mammalian species.     

Adipose-derived stem cell therapy shows promising results for secondary lymphedema

Lymphedema is mainly identified by progressive soft tissue swelling in impaired lymphatic system. Secondary lymphedema attributed to cancer therapy, parasite infection, and trauma remains a serious global disease. Patients with lymphedema suffer swelling, pain, and fatigue, with the dysfunction of the deformed extremities reducing the quality of life and increasing the risk of infection and lymphangiosarcoma. Adipose-derived stem cells (ADSCs) possess prominent regenerative potential to differentiate into multilineage cells, and produce various lymphangiogenic factors, making ADSC therapy a promising approach for lymphedema. The development of lymphedema consists of local inflammation, the fibrosis of lymphatic vessels, and the deposition of adipose fat. Existing animal models do not mimic the chronic inflammation environment, therefore suitable models are required in further studies. Some signal pathways and molecular mechanisms in physiological and pathological lymphagiogenesis remain unclear. In previous animal and human trials, ADSC therapy reduced edema in varying degrees. A larger number of trials with larger samples and longer follow-up periods are required to verify the efficiency and feasibility of ADSC therapy. ADSCs are of easy availability and immune exemption, making them a candidate for lymphedema treatment. Whether ADSCs enhance malignant characteristics or trigger the malignant change deserves further exploration and study before ADSC therapy can be made widely available.     

Dynamic compression and co-culture with nucleus pulposus cells promotes proliferation and differentiation of adipose-derived mesenchymal stem cells

Adipose-derived stem cells (ASCs) are a set of multi potent stem cells potentially used in cartilage tissue engineering. We hypothesized that the effect of dynamic compression and co-culture with nucleus pulposus cells (NPCs) promotes ASC proliferation and chondrogenic differentiation. A controlled dynamic compression loading device was utilized to stimulate ASCs obtained from Sprague Dawley (SD) rats and identified by flow cytometry. The proliferation index was measured by carboxyfluorescein succinimidyl ester (CFSE) staining. Dynamic compression, as well as co-culture enhanced chondrogenic differentiation of ASCs as indicated by the expression of SOX-9, type-II collagen and aggrecan, which were measured by real-time PCR and Western blot. In our study, we found dynamic compression promoted the proliferation of ASCs and induced its differentiation into NP-like cells. Combination of dynamic compression and co-culture showed an additive effect on NP-like cell differentiation.     

成体干细胞的可塑性:横向分化还是细胞融合?

近年来研究显示成体干细胞(adult stem cells)具有可塑性(plasticity),不仅可以生成它们所在组织的成熟细胞,而且在特定环境下能分化成其他组织类型细胞,这种跨系或跨胚层分化现象称为横向分化或转分化(transdifferentiation)。横向分化已为成体干细胞的研究和临床应用包括组织器官损伤的修复提供了新的思路和应用前景。然而,最近的一些研究进展又引出不同的解释,即成体干细胞的可塑性是由于细胞融合(cellfusion)的结果。在此,就成体干细胞的可塑性、横向分化、细胞融合等方面研究作一综述。