Guangxiu Lu

LFA-1 and VLA-4 involved in human high proliferative potentialendothelial progenitor cells homing to ischemic tissue

Cumulative evidences have revealed that endothelial progenitor cell (EPC) transplantation can promote the neovascularization in ischemic tissue, but the mechanism of EPCs homing to the site of ischemia is poorly understood. In this study, to investigate the mechanism of human umbilical cord blood-derived high proliferative potential-endothelial progenitor cells (HPP-EPCs) homing to ischemic tissue we evaluated the expression of lymphocyte function-associated antigen-1 (LFA-1, or CD11a/CD18) and very late antigen-4 (VLA-4, or CD49d/CD29) in EPCs and the changes of expression level of their ligands, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), in ischemic tissue and performed the adhesion and migration assays to analyze the interaction between the receptors and ligands. Furthermore, we studied the roles of LFA-1 and VLA-4 in EPC homing in an ischemic model of mice. The results show that LFA-1 andVLA-4 were expressed in HPPEPCs and ICAM-1 and VCAM-1 were expressed in vessel endothelium in ischemic tissues. The pre-incubation of HPP-EPCs with neutralizing antibodies against CD11a or CD49d reduced adhesion and migration of HPP-EPCs in vitro and reduced recovery of hind-limb blood flow, capillary density and incorporation of HPP-EPC into ischemic tissues in vivo. Furthermore, the pre-incubation of HPP-EPCs with the combination of CD11a and CD49d antibodies led to synergistically negative effects on adhesion and transmigration of HPP-EPCs in vitro, and on the homing of HPP-EPCs to ischemic tissue and on neovascularization capacity in vivo. These results indicate that LFA-1 andVLA-4 are involved in HPP-EPC homing to ischemic tissues.

In vitro hematopoietic differentiation of human embryonic stem cells induced by co-culture with human bone marrow stromal cells and low dose cytokines

Human embryonic stem (hES) cells randomly differentiate into multiple cell types during embryoid body (EB) development and limited studies have focused on directed hematopoietic differentiation. Here, we report that the treatment of hES cells during EBs development with a combination of low dose hematopoietic cytokines, including stem cell factor (SCF), Flt‐3 ligand, vascular endothelial growth factor (VEGF) and human bone marrow stromal cells (hBMSCs), generated cell clusters that contained 8.81% KDR‐positive hemangioblasts, 9.94% CD34‐positive hematopoietic stem cells and 25.7% CD45‐positive mature hematopoietic cells, and expressed hematopoietic genes such as KDR, stem cell leukemia (scl) and runt‐related transcription factor 1 (Runx1). We provide the first evidence for the role of the cytokine—hBMSCs combination in promoting hematopoietic differentiation of hES cells, and thus provide the potential for generation of hematopoietic cells, as well as for understanding early developmental events that govern the initiation of hematopoiesis in humans.

Angiogenic potential difference between two types of endothelial progenitor cells from human umbilical cord blood

The hierarchy of endothelial progenitor cells (EPCs) in human umbilical cord blood has been disclosed. In this study we compare, for the first time, the angiogenic potential difference between two types of EPCs. We cultured mononuclear cells (MNCs) isolated from human umbilical cord blood using endothelial cell‐conditioned medium and obtained two types of EPCs, referred to as circulating angiogenic cells (CACs) and high proliferative potential endothelial progenitor cells (HPP‐EPCs). Both types of cells possess characteristics of EPCs, including expressing CD31, VE‐cadherin, KDR and von Willebrand factor, uptake of Ac‐LDL and binding to lectin. However, unlike CACs, which express CD14 but not CD133, HPP‐EPCs express CD133 but not CD14. Also, unlike CACs, HPP‐EPCs display stronger proliferation and clonogenic potential in vitro and show stronger ability to promote vascular growth in the hind‐limb model of ischemia in mice (BALB/C‐nu) in vivo.

Effect of oxidized low-density lipoprotein on survival and function of endothelial progenitor cell mediated by p38 signal pathway.

This study was designed to investigate whether oxidized low-density lipoprotein (oxLDL) affects the survival and activity of endothelial progenitor cell (EPC) mediated by p38 mitogen-activated protein kinase (MAPK). EPCs were isolated from human peripheral blood in endothelial cell growth medium-2. Incubation with oxLDL at 100 μg/mL decreased EPC number. Treated with oxLDL resulted in increase of EPC apoptosis and in decrease of EPC proliferation. Treatment with oxLDL resulted in a significantly reduced migratory rate of EPCs and reduced adhesion to fibronectin. Treatment with oxLDL impaired the in vitro angiogenesis ability of EPCs. However, all the detrimental effects on EPC were attenuated by pretreatment of EPCs with SB203580, an inhibitor of the p38 MAPK. In addition, the inhibition of the p38-kinase by SB203580 also significantly improved basal number and functions of EPCs. Western blot analysis revealed that oxLDL induced dose- and time-dependent activation of the p38 MAPK. These results demonstrated that p38 MAPK plays a critical role in regulating the number and functions of EPCs in vitro. SB203580 can improve the number and functions of EPCs under basal conditions and prevent the negative effects of oxLDL on the number and functions of EPCs and may be useful to improve the number and function of EPCs for potential cell therapy.

Proliferative feeder cells support prolonged expansion of human embryonic stem cells

Long‐term proliferation of human embryonic stem (hES) cells is currently achieved by co‐culturing with mitotically inactive primary mouse embryonic fibroblasts (mEFs), which serve as feeder cells. This study explores the possibility that proliferative mEFs can be used as feeder cells to maintain the prolonged expansion of hES cells. All undifferentiated hES cell clumps were re‐plated on six different densities of proliferative mEFs. hES colonies cultured on 1 × 105–5 × 105 proliferative mEFs amplified over 130 days of continuous culture and remained undifferentiated, as did those cultured on mitotically inactive mEFs. This suggests that certain densities of proliferative mEFs can maintain the propagation of hES cells, which may be helpful for identifying the cytokines and adhesion molecules that are required for their self‐renewal.

Molecular cloning of a novel rat gene Tsarg1, a member of the DnaJ/HSP40 protein family

Beginning with a mouse gene mTSARG3, which was related to apoptosis of spermatogenic cells, bioinformatics was applied and a predicted novel rat gene full-length cDNA sequence was attained. Gene-specific primers were designed for PCR in rat testis cDNA library. A new gene Tsarg1 (GenBank Accession No. AY380804) was cloned, which is related to apoptosis in rat spermatogenic cells. The gene whose full cDNA length is 1176u2009bp containing 8 exons and 7 introns is located in rat chromosome 1q32–1q33, which encoded a protein containing 316 amino acid residues and being a new member of HSP40 protein family since the sequence contains the highly conserved J domain, which is present in all DnaJ-like proteins and is supported to have a critical role in DnaJ–DnaK protein–protein interactions. The results of RT-PCR and Northern blot analysis showed that Tsarg1 was specifically expressed in rat testis, which probably inhibits rat testis spermatogenic cell apoptosis.

Effects of human yolk sac endothelial cells on supporting expansion of hematopoietic stem/progenitor cells from cord blood

In order to investigate the effects of human yolk sac‐derived endothelial cells (hYSECs) on the expansion of human hematopoietic stem/progenitor cells (HS/PCs) from umbilical cord blood (UCB) in vitro, we purified hYSEC‐like cells from 4–5 week human yolk sacs, which were morphologically similar to endothelial cells and expressed CD31, CD144 and vWF characteristics of endothelial cells. Then we isolated CD34+ cells from UCB in culture under three different conditions: with hematopoietic cytokines (CKs), contact‐coculture or noncontact‐coculture with hYSECs supplemented with CKs, and found that the contact‐coculture system had the strongest expansion efficiency in the total cells (TCs) ability to form HPP‐CFCs. Erythroid burst‐forming units (BFU‐E) increased 52.35‐fold, 20.26‐fold and 27.77‐fold, respectively, compared with pre‐expansion. We detected that the mRNA of Notch ligands such as Jagged1, Delta1 and Delta4 could express in hYSECs after contacted culture with UCB‐CD34+ cells but not the noncontacted cells by RT‐PCR analysis. Therefore, we concluded that the contact‐coculture system supplemented with CKs could support the expansion of UCB‐HS/PCs in vitro, especially high potential proliferative colony‐forming cells (HPP‐CFC) and BFU‐E, perhaps owing to Notch signal pathway.

Characterization and comparison of embryonic stem cell-derived KDR + cells with endothelial cells

Growing interest in utilizing endothelial cells (ECs) for therapeutic purposes has led to the exploration of human embryonic stem cells (hESCs) as a potential source for endothelial progenitors. In this study, ECs were induced from hESC lines and their biological characteristics were analyzed and compared with both cord blood endothelial progenitor cells (CBEPCs) and human umbilical vein endothelial cells (HUVECs) in vitro. The results showed that isolated embryonic KDR+ cells (EC-KDR+) display characteristics that were similar to CBEPCs and HUVECs. EC-KDR+, CBEPCs and HUVECs all expressed CD31 and CD144, incorporated DiI-Ac-LDL, bound UEA1 lectin, and were able to form tube-like structures on Matrigel. Compared with CBEPCs and HUVECs, the expression level of endothelial progenitor cell markers such as CD133 and KDR in EC-KDR+ was significantly higher, while the mature endothelial marker vWF was lowly expressed in EC-KDR+. In summary, the study showed that EC-KDR+ are primitive endothelial-like progenitors and might be a potential source for therapeutic vascular regeneration and tissue engineering.

Effects of an endothelial cell-conditioned medium on the hematopoietic and endothelial differentiation of embryonic stem cells

We have examined the effect of mouse bone marrow endothelial cell‐conditioned medium (mEC‐CM) on hematopoietic and endothelial differentiation of mouse embryonic stem cells (mESCs). mEC‐CM can efficiently promote the differentiation of mESCs into Flk+ cells and hematopoietic colony‐forming cells. mEC‐CM proved to be as potent as a cytokine cocktail comprised of VEGF, bFGF, IGF and EGF. After inducing mESCs with mEC‐CM, cobblestone‐like cells were mechanically selected and identified which had the ability to incorporate DiI‐Ac‐LDL. DiI‐Ac‐LDL‐positive cells were endothelial‐like cells due to their expression of CD31 and Flk1, ability to bind to UEA1 and capacity to form capillary‐like tube structures on matrigel. In conclusion, mEC‐CM can efficiently promote the differentiation of mESCs into endothelial cells and hematopoietic colony‐forming cells. The differentiated endothelial‐like cells can be isolated by using DiI‐Ac‐LDL labeling and mechanical selection.

Preliminary study on human fibroblasts as feeder layer for human embryonic stem cells culturein vitro

To avoid the direct contact with mouse cells and possible heterogeneous pathogen in future application, we need to replace mouse embryonic fibroblasts with human fibroblasts as the feeder layer to maintain human embryonic stem cells growth in the undifferentiated state. We successfully use human fibroblasts derived from aborted fetus and adult prepuce as feeder layer to maintain human embryonic stem cells growth. During the passage and growth on this feeder layer, the human embryonic stem cells can keep their undifferentiated state.