ZNF281 knockdown induced osteogenic differentiation of human multipotent stem cells in vivo and in vitro.

ZNF281 is one of the core transcription factors in embryonic stem cells (ESCs) and has activation and repression roles in the transcription of ESC genes. A known target molecule of Zfp281 (the mouse homologue of ZNF281) is Nanog. However, NANOG is not expressed in most human multipotent stem cells (hMSCs). Here, we investigated the roles of ZNF281 with a gain- and loss-of-function study. The knockdown of ZNF281 in vivo and in vitro resulted in spontaneous osteochondrogenic differentiation and reduced the proliferation of hMSCs, as determined by cell morphology and molecular markers. When ZNF281-knockdown hMSCs were subcutaneously implanted into mice along with ß-tricalcium phosphate (ß-TCP), many cells were converted into osteoblasts within 4 weeks. In contrast, the overexpression of ZNF281 in hMSCs resulted in accelerated proliferation. The expression pattern of ZNF281 correlated well with the expression of ß-CATENIN during differentiation and in the gain/loss-of-function study in hMSCs. The binding of ZNF281 to the promoter region of ß-CATENIN was observed using a chromatin immunoprecipitation (ChIP) assay. In conclusion, we propose that ZNF281 plays an important role in the maintenance and osteogenic differentiation of stem cells via the transcriptional regulation of genes including ß-CATENIN.

The simplest method for in vitro ß-cell production from human adult stem cells.

Diabetes mellitus is a challenging autoimmune disease. Biomedical researchers are currently exploring efficient and effective ways to solve this challenge. The potential of stem cell therapies for treating diabetes represents one of the major focuses of current research on diabetes treatment. Here, we have attempted to differentiate adult stem cells from umbilical cord blood-derived mesenchymal cells (UCB-MSC), Wharton's jelly-derived mesenchymal stem cells (WJ-MSC) and amniotic epithelial stem cells (AE-SC) into insulin-producing cells. The serum-free protocol developed in this study resulted in the differentiation of cells into definitive endoderm, pancreatic foregut, pancreatic endoderm and, finally, pancreatic endocrine cells, which expressed the marker genes SOX17, PDX1, NGN3, NKX6.1, INS, GCG, and PPY, respectively. Detection of the expression of the gap junction-related gene connexin-36 (CX36) using RT-PCR provided conclusive evidence for insulin-producing cell differentiation. In addition to this RT-PCR result, insulin and C-peptide protein were detected by immunohistochemistry and ELISA. Glucose stimulation test results showed that significantly greater amounts of C-peptide and insulin were released from differentiated cells than from undifferentiated cells. In conclusion, the methods investigated in this study can be considered an effective and efficient means of obtaining insulin-producing cells from adult stem cells within a week.

The regulatory role of c-MYC on HDAC2 and PcG expression in human multipotent stem cells.

Myelocytomatosis oncogene (c-MYC) is a well-known nuclear oncoprotein having multiple functions in cell proliferation, apoptosis and cellular transformation. Chromosomal modification is also important to the differentiation and growth of stem cells. Histone deacethylase (HDAC) and polycomb group (PcG) family genes are well-known chromosomal modification genes. The aim of this study was to elucidate the role of c-MYC in the expression of chromosomal modification via the HDAC family genes in human mesenchymal stem cells (hMSCs). To achieve this goal, c-MYC expression was modified by gene knockdown and overexpression via lentivirus vector. Using the modified c-MYC expression, our study was focused on cell proliferation, differentiation and cell cycle. Furthermore, the relationship of c-MYC with HDAC2 and PcG genes was also examined. The cell proliferation and differentiation were checked and shown to be dramatically decreased in c-MYC knocked-down human umbilical cord blood-derived MSCs, whereas they were increased in c-MYC overexpressing cells. Similarly, RT-PCR and Western blotting results revealed that HDAC2 expression was decreased in c-MYC knocked-down and increased in c-MYC overexpressing hMSCs. Database indicates presence of c-MYC binding motif in HDAC2 promoter region, which was confirmed by chromatin immunoprecipitation assay. The influence of c-MYC and HDAC2 on PcG expression was confirmed. This might indicate the regulatory role of c-MYC over HDAC2 and PcG genes. c-MYCs' regulatory role over HDAC2 was also confirmed in human adipose tissue-derived MSCs and bone-marrow derived MSCs. From this finding, it can be concluded that c-MYC plays a vital role in cell proliferation and differentiation via chromosomal modification.

REX-1 expression and p38 MAPK activation status can determine proliferation/differentiation fates in human mesenchymal stem cells.

BACKGROUND: REX1/ZFP42 is a well-known embryonic stem cell (ESC) marker. However, the role of REX1, itself, is relatively unknown because the function of REX1 has only been reported in the differentiation of ESCs via STAT signaling pathways. Human mesenchymal stem cells (hMSCs) isolated from young tissues and cancer cells express REX1. METHODOLOGY/PRINCIPAL FINDING: Human umbilical cord blood-derived MSCs (hUCB-MSCs) and adipose tissue-derived MSCs (hAD-MSCs) strongly express REX1 and have a lower activation status of p38 MAPK, but bone marrow-derived MSCs (hBM-MSCs) have weak REX1 expression and higher activation of p38 MAPK. These results indicated that REX1 expression in hMSCs was positively correlated with proliferation rates but inversely correlated with the phosphorylation of p38 MAPK. In hUCB-MSCs, the roles of REX1 and p38 MAPK were investigated, and a knockdown study was performed using a lentiviral vector-based small hairpin RNA (shRNA). After REX1 knockdown, decreased cell proliferation was observed. In REX1 knocked-down hUCB-MSCs, the osteogenic differentiation ability deteriorated, but the adipogenic potential increased or was similar to that observed in the controls. The phosphorylation of p38 MAPK in hUCB-MSCs significantly increased after REX1 knockdown. After p38 MAPK inhibitor treatment, the cell growth in REX1 knocked-down hUCB-MSCs almost recovered, and the suppressed expression levels of CDK2 and CCND1 were also restored. The expression of MKK3, an upstream regulator of p38 MAPK, significantly increased in REX1 knocked-down hUCB-MSCs. The direct binding of REX1 to the MKK3 gene was confirmed by a chromatin immunoprecipitation (ChIP) assay. CONCLUSIONS/SIGNIFICANCE: These findings showed that REX1 regulates the proliferation/differentiation of hMSCs through the suppression of p38 MAPK signaling via the direct suppression of MKK3. Therefore, p38 MAPK and REX-1 status can determine the cell fate of adult stem cells (ASCs). These results were the first to show the role of REX1 in the proliferation/differentiation of ASCs.

Human umbilical cord blood mesenchymal stem cell-derived extracellular matrix prohibits metastatic cancer cell MDA-MB-231 proliferation.

It is not clear whether adult stem cell extracellular matrix (ECM) can regulate cancer cells. We demonstrated that the ECM produced by UCB-MSCs was able to arrest the growth of metastatic tumor cells by upregulating levels of PTEN in aggressive cancer cells. Human UCB-MSCs produced dickkopf (DKK1) are capable of inhibiting cancer cell proliferation but has no contribution to the tumor inhibition effect of UCB-MSC ECM. This study also provides an innovative approach to specifically examine the effect of stem cell microenvironments on cancer cells without the complexity of cell-cell interactions. In conclusion, human UCB-MSC ECM prohibits cancer cell proliferation.

OCT4A contributes to the stemness and multi-potency of human umbilical cord blood-derived multipotent stem cells (hUCB-MSCs).

The OCT4A gene, a POU homeodomain transcription factor, has been shown to be expressed in embryonic stem cells (ESC) as well as hUCB-MSCs. In this study, the roles played by OCT4A in hUCB-MSCs were determined by stably inhibiting OCT4A with lenti-viral vector-based small hairpin RNA (shRNA). A decreased rate of cell proliferation was observed in OCT4-inhibited hUCB-MSCs. Down-regulation of CCNA2 expression in OCT4-inhibited hUCB-MSCs was confirmed by RT-PCR and real-time RT-PCR analysis in three genetically independent hUCB-MSC clones. Adipogenic differentiation was also suppressed in OCT4-inhibited hUCB-MSCs. The up-regulation of DTX1 and down-regulation of HDAC1, 2, and 4 expressions may be related to this differentiation deformity. The expression of other transcription factors, including SOX2, REX1 and c-MYC, was also affected by OCT4 inhibition in hUCB-MSCs. In conclusion, these finding suggest that OCT4A performs functionally conserved roles in hUCB-MSCs, making its expression biologically important for ex vivo culture of hUCB-MSCs.