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1.  Benefits of hypoxic culture on bone marrow multipotent stromal cells 
Cultivation of cells is usually performed under atmospheric oxygen tension; however, such a condition does not replicate the hypoxic conditions of normal physiological or pathological status in the body. Recently, the effects of hypoxia on bone marrow multipotent stromal cells (MSCs) have been investigated. In a long-term culture, hypoxia can inhibit senescence, increase the proliferation rate and enhance differentiation potential along the different mesenchymal lineages. Hypoxia also modulates the paracrine effects of MSCs, causing upregulation of various secretable factors, including the vascular endothelial growth factor and interleukin-6, and thereby promoting wound healing and diabetic fracture healing. Finally, hypoxia plays an important role in mobilization and homing of MSCs, primarily by its ability to induce stromal cell-derived factor-1 expression along with its receptor, CXCR4. After transplantation, an ischemic environment, that is the combination of hypoxia and lack of nutrition, can lead to apoptosis or cell death, which can be overcome by the hypoxic preconditioning of MSCs and overexpression of prosurvival genes like Akt, HO-1 and Hsp70. This review emphasizes that hypoxia is an important factor in all major aspects of stem cell biology, and the mechanism involved in the hypoxic inducible factor-1signaling pathway behind these responses is also discussed.
PMCID: PMC3484415  PMID: 23119226
Mesenchymal stem cells; hypoxia; hypoxic preconditioning; proliferation; differentiation potential; apoptosis; migration; engraftment; HIF-1
2.  Functional roles of pluripotency transcription factors in mesenchymal stem cells 
Cell Cycle  2012;11(20):3711-3712.
PMCID: PMC3495805  PMID: 22951581
mesenchymal stem cells; pluripotency transcription factor; Oct4; Nanog; Dnmt1; self-renewal; proliferation; spontaneous differentiation
3.  Twist Controls Skeletal Development and Dorsoventral Patterning by Regulating Runx2 in Zebrafish 
PLoS ONE  2011;6(11):e27324.
Twist1a and twist1b are the principal components of twists that negatively regulate a number of cellular signaling events. Expression of runx2 and downstream targets is essential for skeletal development and ventral organizer formation and specification in early vertebrate embryos, but what controls ventral activity of maternal runx2 and how twists function in zebrafish embryogenesis still remain unclear.
Methodology/Principal Findings
By studying the loss of twist induced by injection of morpholino-oligonucleotide in zebrafish, we found that twist1a and twist1b, but not twist2 or twist3, were required for proper skeletal development and dorsoventral patterning in early embryos. Overexpression of twist1a or twist1b following mRNA injection resulted in deteriorated skeletal development and formation of typical dorsalized embryos, whereas knockdown of twist1a and twist1b led to the formation of abnormal embryos with enhanced skeletal formation and typical ventralized patterning. Overexpression of twist1a or twist1b decreased the expression of runx2b, whereas twist1a and twist1b knockdown increased runx2b expression. We have further demonstrated that phenotypes induced by twist1a and twist1b knockdown were rescued by runx2b knockdown.
Together, these results suggest that twist1a and twist1b control skeletal development and dorsoventral patterning by regulating runx2b in zebrafish and provide potential targets for the treatment of diseases or syndromes associated with decreased skeletal development.
PMCID: PMC3210159  PMID: 22087291
4.  Hypoxia Inhibits Osteogenesis in Human Mesenchymal Stem Cells through Direct Regulation of RUNX2 by TWIST 
PLoS ONE  2011;6(9):e23965.
Bone loss induced by hypoxia is associated with various pathophysiological conditions, however, little is known about the effects of hypoxia and related signaling pathways on osteoblast differentiation and bone formation. Because bone marrow-derived mesenchymal stem cells (MSCs) survive under hypoxic conditions and readily differentiate into osteoblasts by standard induction protocols, they are a good in vitro model to study the effects of hypoxia on osteoblast differentiation.
Methodology/Principle Findings
Using human MSCs, we discovered TWIST, a downstream target of HIF-1╬▒, was induced under hypoxia and acted as a transcription repressor of RUNX2 through binding to the E-box located on the promoter of type 1 RUNX2. Suppression of type 1 RUNX2 by TWIST under hypoxia further inhibited the expression of BMP2, type 2 RUNX2 and downstream targets of RUNX2 in MSCs.
Our findings point to the important role of hypoxia-mediated signalling in osteogenic differentiation in MSCs through direct regulation of RUNX2 by TWIST, and provide a method for modifying MSC osteogenesis upon application of these cells in fracture healing and bone reconstruction.
PMCID: PMC3170288  PMID: 21931630
5.  Overexpression of hTERT increases stem-like properties and decreases spontaneous differentiation in human mesenchymal stem cell lines 
To overcome loss of stem-like properties and spontaneous differentiation those hinder the expansion and application of human mesenchymal stem cells (hMSCs), we have clonally isolated permanent and stable human MSC lines by ectopic overexpression of primary cell cultures of hMSCs with HPV 16 E6E7 and human telomerase reverse transcriptase (hTERT) genes. These cells were found to have a differentiation potential far beyond the ordinary hMSCs. They expressed trophoectoderm and germline specific markers upon differentiation with BMP4 and retinoic acid, respectively. Furthermore, they displayed higher osteogenic and neural differentiation efficiency than primary hMSCs or hMSCs expressed HPV16 E6E7 alone with a decrease in methylation level as proven by a global CpG island methylation profile analysis. Notably, the demethylated CpG islands were highly associated with development and differentiation associated genes. Principal component analysis further pointed out the expression profile of the cells converged toward embryonic stem cells. These data demonstrate these cells not only are a useful tool for the studies of cell differentiation both for the mesenchymal and neurogenic lineages, but also provide a valuable source of cells for cell therapy studies in animal models of skeletal and neurological disorders.
PMCID: PMC2923118  PMID: 20670406
6.  Fibronectin and laminin promote differentiation of human mesenchymal stem cells into insulin producing cells through activating Akt and ERK 
Islet transplantation provides a promising cure for Type 1 diabetes; however it is limited by a shortage of pancreas donors. Bone marrow-derived multipotent mesenchymal stem cells (MSCs) offer renewable cells for generating insulin-producing cells (IPCs).
We used a four-stage differentiation protocol, containing neuronal differentiation and IPC-conversion stages, and combined with pellet suspension culture to induce IPC differentiation.
Here, we report adding extracellular matrix proteins (ECM) such as fibronectin (FN) or laminin (LAM) enhances pancreatic differentiation with increases in insulin and Glut2 gene expressions, proinsulin and insulin protein levels, and insulin release in response to elevated glucose concentration. Adding FN or LAM induced activation of Akt and ERK. Blocking Akt or ERK by adding LY294002 (PI3K specific inhibitor), PD98059 (MEK specific inhibitor) or knocking down Akt or ERK failed to abrogate FN or LAM-induced enhancement of IPC differentiation. Only blocking both of Akt and ERK or knocking down Akt and ERK inhibited the enhancement of IPC differentiation by adding ECM.
These data prove IPC differentiation by MSCs can be modulated by adding ECM, and these stimulatory effects were mediated through activation of Akt and ERK pathways.
PMCID: PMC2915967  PMID: 20624296

Results 1-6 (6)