The balance of positive and negative effects of TGF-ß signaling regulates the development of hematopoietic and endothelial progenitors in human pluripotent stem cells.

Derived from mesoderm precursors, hemangioblasts are bipotential common progenitors of hematopoietic cells and endothelial cells. The regulatory events controlling hematopoietic and endothelial lineage specification are largely unknown, especially in humans. In this study, we establish a serum-free and feeder-free system with a high-efficient embryoid body (EB) generation to investigate the signals that direct differentiation of human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs). Consistent with previous studies, the CD34(+)CD31(+)VE-cadherin(+) (VEC(+)) cells derived from hPSCs contain hematopoietic and endothelial progenitors. In the presence of hematopoietic and endothelial growth factors, some of CD34(+)CD31(+)VEC(+) cells give rise to blast colony-forming cells (BL-CFCs), which have been used to characterize bipotential hemangioblasts. We found that the level of the transforming growth factor beta (TGF-ß) 1 protein is increased during hPSC differentiation, and that TGF-ß signaling has the double-edged effect on hematopoietic and endothelial lineage differentiation in hPSCs. An addition of TGF-ß to hPSC differentiation before mesoderm induction promotes the development of mesoderm and the generation of CD34(+)CD31(+)VEC(+) cells. An addition of TGF-ß inhibitor, SB431542, before mesoderm induction downregulates the expression of mesodermal markers and reduces the number of CD34(+)CD31(+)VEC(+) progenitor cells. However, inhibition of TGF-ß signaling after mesoderm induction increases CD34(+)CD31(+)VEC(+) progenitors and BL-CFCs. These data provide evidence that a balance of positive and negative effects of TGF-ß signaling at the appropriate timing is critical, and potential means to improve hematopoiesis and vasculogenesis from hPSCs.

[Preparation and identification of monoclonal antibody against human Flt-1].

AIM: To prepare functional monoclonal antibodies(mAb)against recombinant human Flt-1(rhFlt-1). METHODS: A cell line stable secreting mAb was established by using FLT-1 extracellular domain III as antigen and hybridoma technique. Then it was purified in large-scale from mouse ascites by protein G affinity chromatography. The characteristics of mAb were then determined by ELISA, Western blotting and FACS. RESULTS: The immunoglobin subtype of mAb XA12 was IgG1 with kappa (κ) light chains, and it could recognize rhFlt-1 specifically. Furthermore, mAb XA12 could bind to rhFlt-1with high affinity (K=1.28±0.05 nmol/L). It could also be used to detect Flt-1-positive cells, such as human umbilical vascular endothelial cells (HUVECs) and K562/A02 in a dose-dependent fashion. CONCLUSION: A hybridoma cell line secreting functional anti-rhFlt-1 mAb was successfully prepared. The antibody can be used to study the function of Flt-1 and further potentially optimized for clinical purpose.

Bcl-xL enhances single-cell survival and expansion of human embryonic stem cells without affecting self-renewal.

Robust expansion and genetic manipulation of human embryonic stem cells (hESCs) and induced-pluripotent stem (iPS) cells are limited by poor cell survival after enzymatic dissociation into single cells. Although inhibition of apoptosis is implicated for the single-cell survival of hESCs, the protective role of attenuation of apoptosis in hESC survival has not been elucidated. Bcl-xL is one of several anti-apoptotic proteins, which are members of the Bcl-2 family of proteins. Using an inducible system, we ectopically expressed Bcl-xL gene in hESCs, and found a significant increase of hESC colonies in the single-cell suspension cultures. Overexpression of Bcl-xL in hESCs decreased apoptotic caspase-3(+) cells, suggesting attenuation of apoptosis in hESCs. Without altering the kinetics of pluripotent gene expression, the efficiency to generate embryoid bodies (EBs) in vitro and the formation of teratoma in vivo were significantly increased in Bcl-xL-overexpressing hESCs after single-cell dissociation. Interestingly, the number and size of hESC colonies from cluster cultures were not affected by Bcl-xL overexpression. Several genes of extracellular matrix and adhesion molecules were upregulated by Bcl-xL in hESCs without single-cell dissociation, suggesting that Bcl-xL regulates adhesion molecular expression independent of cell dissociation. In addition, the gene expressions of FAS and several TNF signaling mediators were downregulated by Bcl-xL. These data support a model in which Bcl-xL promotes cell survival and increases cloning efficiency of dissociated hESCs without altering hESC self-renewal by i) attenuation of apoptosis, and ii) upregulation of adhesion molecules to facilitate cell-cell or cell-matrix interactions.

[Effects of catalase on human umbilical cord mesenchymal stem cells].

This study was aimed to investigate the growth and multiple differentiation potential of human umbilical cord tissue derived mesenchymal stem cells (UC-MSCs) transfected by a retroviral vector with catalase (CAT) gene. The UC-MSCs cultured in vitro were transfected by using pMSCV carrying GFP (pMSCV-GFP) and pMSCV carrying CAT (pMSCV-GFP-CAT) respectively, then the MSC-GFP cell line and MSC-GFP-CAT cell line were obtained by sorting of flow cytometry. The GFP expression was observed by a fluorescent microscopy at 48 hours after CAT gene transfection. The GFP+ cells were sorted by flow cytometry. The activity of CAT in GFP+ cells was detected by catalase assay kit. The proliferative capacity of transfected UC-MSCs was determined by cell counting kit-8. The differentiation ability of gene-transfected GFP+ cells into osteogenesis and adipogenesis was observed by von Kossa and oil red O staining. The results indicated that green fluorescence in UC-MSCs was observed at 48 hours after transfection, and the fluorescence gradually enhanced to a steady level on day 3. The percentage of MSCs-GFP was (25.54+/-8.65)%, while the percentage of MSCs-GFP-CAT was (35.4+/-18.57)%. The activity of catalase in UC-MSCs, MSCs-GFP, MSCs-GFP-CAT cells were 19.5, 20.3, 67.2 U, respectively. The transfected MSCs-GFP-CAT could be induced into osteoblasts and adipocytes. After 21 days, von Kossa staining showed induced osteoblasts. Many lipid droplets with high refractivity occurred in cytoplasm of the transfected UC-MSCs, and showed red fat granules in oil red O staining cells. There were no significant differences between transfected and non-transfected UC-MSCs cells (p>0.05). It is concluded that UC-MSCs are successfully transfected by retrovirus carrying GFP or CAT gene, the activity of catalase increased by 3.4-fold. The transfected UC-MSCs maintain proliferation potential and ability of differentiation into osteoblasts and adipocytes.