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.     

bFGF promotes adipocyte differentiation in human mesenchymal stem cells derived from embryonic stem cells

In this work we describe the establishment of mesenchymal stem cells (MSCs) derived from embryonic stem cells (ESCs) and the role of bFGF in adipocyte differentiation. The totipotency of ESCs and MSCs was assessed by immunofluorescence staining and RT-PCR of totipotency factors. MSCs were successfully used to induce osteoblasts, chondrocytes and adipocytes. MSCs that differentiated into adipocytes were stimulated with and without bFGF. The OD/DNA (optical density/content of total DNA) and expression levels of the specific adipocyte genes PPARγ2 (peroxisome proliferator activated receptor γ2) and C/EBPs were higher in bFGF cells. Embryonic bodies had a higher adipocyte level compared with cells cultured in plates. These findings indicate that bFGF promotes adipocyte differentiation. MSCs may be useful cells for seeding in tissue engineering and have enormous therapeutic potential for adipose tissue engineering.     

Physiologically based microenvironment for in vitro neural differentiation of adipose-derived stem cells

The limited capacity of nervous system to promote a spontaneous regeneration and the high rate of neurodegenerative diseases appearance are keys factors that stimulate researches both for defining the molecular mechanisms of pathophysiology and for evaluating putative strategies to induce neural tissue regeneration. In this latter aspect, the application of stem cells seems to be a promising approach, even if the control of their differentiation and the maintaining of a safe state of proliferation should be troubled. Here, we focus on adipose tissue-derived stem cells and we seek out the recent advances on the promotion of their neural differentiation, performing a critical integration of the basic biology and physiology of adipose tissuederived stem cells with the functional modifications that the biophysical, biomechanical and biochemical microenvironment induces to cell phenotype. The pre-clinical studies showed that the neural differentiation by cell stimulation with growth factors benefits from the integration with biomaterials and biophysical interaction like microgravity. All these elements have been reported as furnisher of microenvironments with desirable biological, physical and mechanical properties. A critical review of current knowledge is here proposed, underscoring that a real advance toward a stable, safe and controllable adipose stem cells clinical application will derive from a synergic multidisciplinary approach that involves material engineer, basic cell biology, cell and tissue physiology.     

Trafficking and differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are a heterogeneous population of stem/progenitor cells with pluripotent capacity to differentiate into mesodermal and non‐mesodermal cell lineages, including osteocytes, adipocytes, chondrocytes, myocytes, cardiomyocytes, fibroblasts, myofibroblasts, epithelial cells, and neurons. MSCs reside primarily in the bone marrow, but also exist in other sites such as adipose tissue, peripheral blood, cord blood, liver, and fetal tissues. When stimulated by specific signals, these cells can be released from their niche in the bone marrow into circulation and recruited to the target tissues where they undergo in situ differentiation and contribute to tissue regeneration and homeostasis. Several characteristics of MSCs, such as the potential to differentiate into multiple lineages and the ability to be expanded ex vivo while retaining their original lineage differentiation commitment, make these cells very interesting targets for potential therapeutic use in regenerative medicine and tissue engineering. The feasibility for transplantation of primary or engineered MSCs as cell‐based therapy has been demonstrated. In this review, we summarize the current knowledge on the signals that control trafficking and differentiation of MSCs. J. Cell. Biochem. 106: 984–991, 2009. © 2009 Wiley‐Liss, Inc.     

Current applications of adipose-derived stem cells and their future perspectives

Adult stem cells have a great potential to treat various diseases. For these cell-based therapies, adipose-derived stem cells(ADSCs) are one of the most promising stem cell types, including embryonic stem cells(ESCs) and induced pluripotent stem cells(iPSCs). ESCs and iPSCs have taken center stage due to their pluripotency. However, ESCs and iPSCs have limitations in ethical issues and in identification of characteristics, respectively. Unlike ESCs and iPSCs, ADSCs do not have such limitations and are not only easily obtained but also uniquely expandable. ADSCs can differentiate into adipocytes, osteoblasts, chondrocytes, myocytes and neurons under specific differentiation conditions, and these kinds of differentiation potential of ADSCs could be applied in regenerative medicine e.g., skin reconstruction, bone and cartilage formation, etc. In this review, the current status of ADSC isolation, differentiation and their therapeutic applications are discussed.     

Current applications of adipose-derived stem cells and their future perspectives简

Adult stem cells have a great potential to treat various diseases. For these cell-based therapies, adipose-derived stem cells (ADSCs) are one of the most promising stem cell types, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). ESCs and iPSCs have taken center stage due to their pluripotency. However, ESCs and iPSCs have limitations in ethical issues and in identification of characteristics, respectively. Unlike ESCs and iPSCs, ADSCs do not have such limitations and are not only easily obtained but also uniquely expandable. ADSCs can differentiate into adipocytes, osteoblasts, chondrocytes, myocytes and neurons under specific differentiation conditions, and these kinds of differentiation potential of ADSCs could be applied in regenerative medicine e.g., skin reconstruction, bone and cartilage formation, etc. In this review, the current status of ADSC isolation, differentiation and their therapeutic applications are discussed.     

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.     

Culture and neural differentiation of rat bone marrow mesenchymal stem cells in vitro

Adult bone marrow mesenchymal stem cells (MSCs) can differentiate into several types of mesenchymal cells, including osteocytes, chondrocytes, and adipocytes, but can also differentiate into non-mesenchymal cells, such as neural cells, under appropriate experimental conditions. Until now, many protocols for inducing neuro-differentiation in MSCs in vitro have been reported. But due to the differences in MSCs' isolation and culture conditions, the results of previous studies lacked consistency and comparability. In this study, we induced differentiation into neural phenotype in the same MSCs population by three different treatments: beta-mercaptoethanol, serum-free medium and co-cultivation with fetal mouse brain astrocytes. In all of the three treatments, MSCs could express neural markers such as NeuN or GFAP, associating with remarkable morphological modifications. But these treatments led to neural phenotype in a non-identical manner. In serum-free medium, MSCs mainly differentiated into neuron-like cells, expressing neuronal marker NeuN, and BME can promote this process. Differently, after co-culturing with astrocytes, MSCs leaned to differentiate into GFAP(+) cells. These data confirmed that MSCs can exhibit plastic neuro-differentiational potential in vitro, depending on the protocols of inducement.     

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.     

Matrix remodeling associated 7 promotes differentiation of bone marrow mesenchymal stem cells toward osteoblasts

The matrix remodeling associated 7 (MXRA7) gene had been ill-studied and its biology remained to be discovered. Inspired by our previous findings and public datasets concerning MXRA7, we hypothesized that the MXRA7 gene might be involved in bone marrow mesenchymal stem cells (BMSCs) functions related to bone formation, which was checked by utilizing in vivo or in vitro methodologies. Micro-computed tomography of MXRA7-deficient mice demonstrated retarded osteogenesis, which was reflected by shorter femurs, lower bone mass in both trabecular and cortical bones compared with wild-type (WT) mice. Histology confirmed the osteopenia-like feature including thinner growth plates in MXRA7-deficient femurs. Immunofluorescence revealed less osteoblasts in MXRA7-deficient femurs. Polymerase chain reaction or western blot analysis showed that when WT BMSCs were induced to differentiate toward osteoblasts or adipocytes in culture, MXRA7 messenger RNA or protein levels were significantly increased alongside osteoblasts induction, but decreased upon adipocytes induction. Cultured MXRA7-deficient BMSCs showed decreased osteogenesis upon osteogenic differentiation induction as reflected by decreased calcium deposition or lower expression of genes responsible for osteogenesis. When recombinant MXRA7 proteins were supplemented in a culture of MXRA7-deficient BMSCs, osteogenesis or gene expression was fully restored. Upon osteoblast induction, the level of active β-catenin or phospho-extracellular signal-regulated kinase in MXRA7-deficient BMSCs was decreased compared with that in WT BMSCs, and these impairments could be rescued by recombinant MXRA7 proteins. In adipogenesis induction settings, the potency of MXRA7-deficient BMSCs to differentiate into adipocytes was increased over the WT ones. In conclusion, this study demonstrated that MXRA7 influences bone formation via regulating the balance between osteogenesis and adipogenesis in BMSCs.     

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.     

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.     

Review of biophysical factors affecting osteogenic differentiation of human adult adipose-derived stem cells

Developing bone is subject to the control of a broad variety of influences in vivo. For bone repair applications, in vitro osteogenic assays are routinely used to test the responses of bone-forming cells to drugs, hormones, and biomaterials. Results of these assays are used to predict the behavior of bone-forming cells in vivo. Stem cell research has shown promise for enhancing bone repair. In vitro osteogenic assays to test the bone-forming response of stem cells typically use chemical solutions. Stem cell in vitro osteogenic assays often neglect important biophysical cues, such as the forces associated with regular weight-bearing exercise, which promote bone formation. Incorporating more biophysical cues that promote bone formation would improve in vitro osteogenic assays for stem cells. Improved in vitro osteogenic stimulation opens opportunities for “pre-conditioning” cells to differentiate towards the desired lineage. In this review, we explore the role of select biophysical factors—growth surfaces, tensile strain, fluid flow and electromagnetic stimulation—in promoting osteogenic differentiation of stem cells from human adipose. Emphasis is placed on the potential for physical microenvironment manipulation to translate tissue engineering and stem cell research into widespread clinical usage.     

Neural differentiation of adipose-derived stem cells isolated from GFP transgenic mice

Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, we have recently reported that adipose-derived stromal cells (ASCs) could differentiate into mesenchymal lineages in vitro. In this study, we performed neural induction using ASCs from GFP transgenic mice and were able to induce these ASCs into neuronal and glial cell lineages. Most of the neurally induced cells showed bipolar or multipolar appearance morphologically and expressed neuronal markers. Electron microscopy revealed their neuronal morphology. Some cells also showed glial phenotypes, as shown immunocytochemically. The present study clearly shows that ASCs derived from GFP transgenic mice differentiate into neural lineages in vitro, suggesting that these cells might provide an ideal source for further neural stem cell research with possible therapeutic application for neurological disorders.     

Pluripotent stem cell-derived neural stem cells: From basic research to applications

Basic research on pluripotent stem cells is designed to enhance understanding of embryogenesis, whereas applied research is designed to develop novel therapies and prevent diseases. Attainment of these goals has been enhanced by the establishment of embryonic stem cell lines, the technological development of genomic reprogramming to generate induced-pluripotent stem cells, and improvements in vitro techniques to manipulate stem cells. This review summarizes the techniques required to generate neural cells from pluripotent stem cells. In particular, this review describes current research applications of a simple neural differentiation method, the neural stem sphere method, which we developed.