Hypoxia inhibits senescence and maintains mesenchymal stem cell properties through down-regulation of E2A-p21 by HIF-TWIST.

Although low-density culture provides an efficient method for rapid expansion of human mesenchymal stem cells (MSCs), MSCs enriched by this method undergo senescence and lose their stem cell properties, which could be preserved by combining low-density and hypoxic culture. The mechanism was mediated through direct down-regulation of E2A-p21 by the hypoxia-inducible factor-1α (HIF-1α)-TWIST axis. Expansion under normoxia induced E2A and p21 expression, which were abrogated by overexpression of TWIST, whereas siRNA against TWIST up-regulated E2A and p21 in hypoxic cells. Furthermore, siRNA against p21 in normoxic cells enhanced proliferation and increased differentiation potential, whereas overexpression of p21 in hypoxic cells induced a decrease in proliferation and a loss of differentiation capacity. More importantly, MSCs expanded under hypoxic conditions by up to 100 population doublings, exhibited telomerase activity with maintained telomere length, normal karyotyping, and intact genetic integrity, and did not form tumors. These results support low-density hypoxic culture as a method for efficiently expanding MSCs without losing stem cell properties or increasing tumorigenicity.

Mesenchymal stem cells promote formation of colorectal tumors in mice.

BACKGROUND & AIMS: Tumor-initiating cells are a subset of tumor cells with the ability to form new tumors; however, they account for less than 0.001% of the cells in colorectal or other types of tumors. Mesenchymal stem cells (MSCs) integrate into the colorectal tumor stroma; we investigated their involvement in tumor initiation. METHODS: Human colorectal cancer cells, MSCs, and a mixture of both cell types were injected subcutaneously into immunodeficient mice. We compared the ability of each injection to form tumors and investigated the signaling pathway involved in tumor initiation. RESULTS: A small number (≤ 10) of unsorted, CD133⁻, CD166⁻, epithelial cell adhesion molecule⁻(EpCAM⁻), or CD133⁻/CD166⁻/EpCAM⁻ colorectal cancer cells, when mixed with otherwise nontumorigenic MSCs, formed tumors in mice. Secretion of interleukin (IL)-6 by MSCs increased the expression of CD133 and activation of Janus kinase 2-signal transducer and activator of transcription 3 (STAT3) in the cancer cells, and promoted sphere and tumor formation. An antibody against IL-6 or lentiviral-mediated transduction of an interfering RNA against IL-6 in MSCs or STAT3 in cancer cells prevented the ability of MSCs to promote sphere formation and tumor initiation. CONCLUSIONS: IL-6, secreted by MSCs, signals through STAT3 to increase the numbers of colorectal tumor-initiating cells and promote tumor formation. Reagents developed to disrupt this process might be developed to treat patients with colorectal cancer.

Apoptosis in chondrogenesis of human mesenchymal stem cells: effect of serum and medium supplements.

Apoptosis is an inevitable process during development and is evident in the formation of articular cartilage and endochondral ossification of growth plate. Mesenchymal stem cells (MSCs) can serve as alternative sources for cell therapy in focal chondral lesions or diffuse osteoarthritis. But there are few, if any, studies investigating apoptosis during chondrogenesis by MSCs. The aim of this study was to find the better condition to prevent apoptosis during chondrogenesis by MSCs. Apoptosis were evaluated in MSCs induced in different chondrogenic media by the use of Annexin V, TUNEL staining, lysosomal labeling with lysotracker and immunostaining of apoptotic markers. We found apparent apoptosis was demonstrated by Annexin V, TUNEL staining and lysosomal labeling during chondrogenesis. Meanwhile, the degree of apoptosis was related to the reagents of the defined chondrogenic medium. Adding serum in medium increased apoptosis, however, TGF-beta1 inhibited apoptosis. The apoptosis was associated with the activation of caspase-3, the increase in the Bax/Bcl-2 ratio, the loss of lysosomal integrity, and the increase of PARP-cleavage. Pro-inflammatory cytokines, IL-1alpha, IL-1beta and TNFalpha did not induce any increase in apoptosis. Interestingly, the inhibition of apoptosis by serum free medium supplemented with ITS was also associated with an increase in the expression of type II collagen, and a decrease in the expression of type X collagen, Runx2, and other osteogenic genes, while TGF-beta1 increased the expression of Sox9, type II and type X collagen and decreased the expression of osteogenic genes. These data suggest apoptosis occurs during chondrogenesis by MSCs by cell death intrinsic pathway activation and this process may be modulated by culture conditions.

Fibronectin and laminin promote differentiation of human mesenchymal stem cells into insulin producing cells through activating Akt and ERK.

BACKGROUND: 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). METHODS: We used a four-stage differentiation protocol, containing neuronal differentiation and IPC-conversion stages, and combined with pellet suspension culture to induce IPC differentiation. RESULTS: 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. CONCLUSIONS: 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.

Mesenchymal stem cell-conditioned medium facilitates angiogenesis and fracture healing in diabetic rats.

The most critical factor for fracture union is the blood supply to the fracture site, which is usually impaired in patients with diabetes. Recently, mesenchymal stem cells-derived conditioned medium (MSC-CM) has shown significantly higher levels of angiogenic factors, such as VEGF and IL-6. We demonstrate in this report that MSC-CM delivered in gelatin sponges stimulates angiogenesis and promotes fracture healing in a diabetic rat model. Subcutaneous implantation of gelatin sponges soaked in MSC-CM demonstrated better tissue ingrowth and higher capillary densities at 2 and 3 weeks than gelatin sponges in minimal essential medium (MEM) or 293 cell-derived conditioned medium (293-CM). Implantation of fibular defects with gelatin sponges soaked in MSC-CM enhanced bone ingrowth and fracture healing rates compared to 293-CM and MEM groups at 8 weeks. Micro-computed tomography analysis further indicated a higher new bone volume in the MSC-CM group compared to the other diabetic groups. Histological analysis with CD31 immunostaining also revealed that MSC-CM increased endothelial cell counts compared to the other groups. Together, these results indicated that gelatin sponges used to deliver MSC-CM promote angiogenesis and fracture healing in a diabetic model and may be an alternative strategy for treating fracture non-union in patients with diabetes.

Isolation of mesenchymal stem cells from human ligamentum flavum: implicating etiology of ligamentum flavum hypertrophy.

STUDY DESIGN: To demonstrate the existence of mesenchymal stem cells (MSCs) in ligamentum flavum (LF) and their pathogenic role in LF hypertrophy. OBJECTIVE: To isolate and characterize LF-derived MSCs and their response to transforming growth factor-beta 1 (TGF-ß1) and trichostatin A (TSA), a histone deacetylase inhibitor (HDACi). SUMMARY OF BACKGROUND DATA: LF is a connective tissue, of which hypertrophic changes induce spinal stenosis. The pathogenic role of TGF-ß1 in spinal stenosis has been implicated. TSA has been shown to suppress TGF-ß1-induced alpha-smooth muscle actin (α-SMA), type I and III collagen synthesis in a variety of cells. MSCs have been isolated from a variety of adult tissues, except LF. Whether MSCs exist in LF and their response to TGF-ß1 and TSA is not clear. METHODS: The MSCs from LF were isolated and cultured. Their phenotypic character, linage differentiation potential, and response to TGF-ß1 and TSA were analyzed. RESULTS: LF-derived MSCs have the similar profile of surface markers as bone marrow MSCs. They were demonstrated to have the potential to be differentiated into osteoblasts, adipocytes, and chondrocytes. Administration of TGF-ß1 stimulated cell proliferation, enhanced the gene expression of type I and III collagen, and increased the gene expression and protein level of α-SMA. TSA blocked the fibrogenic effects of TGF-ß1. CONCLUSION: The current results demonstrated the isolation of MSCs from LF. The cellular response to TGF-ß1 implied that these cells might play an important role in the pathogenesis of LF hypertrophy. TSA, which blocks the effects of TGF-ß1, may be a potent therapeutic choice for inhibiting LF hypertrophy.

Enhancement of wound healing by human multipotent stromal cell conditioned medium: the paracrine factors and p38 MAPK activation.

Wound healing can be improved by transplanting mesenchymal stem cells (MSCs). In this study, we have demonstrated the benefits of the conditioned medium derived from human MSCs (CM-MSC) in wound healing using an excisional wound model. CM-MSC accelerated wound closure with increased reepithelialization, cell infiltration, granulation formation, and angiogenesis. Notably, CM-MSC enhanced epithelial and endothelial cell migration, suggesting the contribution of increased cell migration to wound healing enhanced by CM-MSC. Cytokine array, ELISA analysis, and quantitative RT-PCR revealed high levels of IL-6 in CM-MSC. Moreover, IL-6 added to the preconditioned medium enhanced both cell migration and wound healing, and antibodies against IL-6 blocked the increase in cell motility and wound closure by CM-MSC. The IL-6 secretory pathway of MSCs was inhibited by SB203580, an inhibitor of p38 MAPK or siRNA against p38 MAPK, suggesting IL-6 secretion by MSCs is mediated through the activation of p38 MAPK. Inactivation of p38 MAPK also reduced the expression and production of IL-8 and CXCL1 by MSCs, both of which were also demonstrated to enhance cell migration and wound closure. Thus, our data suggest MSCs promote wound healing through releasing a repertoire of paracrine factors via activation of p38 MAPK, and the CM-MSC may be applied to enhance wound healing.

Genistein inhibits the inward rectifying potassium current in guinea pig ventricular myocytes.

Genistein is an isoflavone with potent inhibitory activity on protein tyrosine kinase. Previous studies have shown that genistein has additional effects, among which the direct blocking effects on various ionic channels have recently been disclosed. Using whole-cell voltage clamp and current clamp techniques, we demonstrate that micromolar concentrations of genistein dose-dependently and reversibly inhibit the inward rectifying K(+) current, and depolarize the resting membrane potential, resulting in abnormal automaticity in guinea pig ventricular myocytes. Interestingly, another potent tyrosine kinase inhibitor, tyrphostin 51, did not produce the same inhibitory effect, while the inactive analogue of genistein, daidzein, had a similar blocking effect. We suggest that genistein directly blocks the inward rectifying K(+) current in ventricular myocytes, and one should be cautious of its pro-arrhythmic effect in clinical use.