Effects of human aorta-gonad-mesonephros region stromal cells on inducing differentiation of murine embryonic stem cells into hematopoietic stem/progenitor cells in vitro / 中国实验血液学杂志

This study was aimed to investigate the effect of human aorta-gonad-mesonephros (AGM) region stromal cells on differentiation of murine embryonic stem cells (ESC) into hematopoietic stem cells (HSC) in vitro and to clarify their effect mechanism. E14 murine ESC were induced into embryo body (EB) firstly. Then the EB cells were further co-cultured with the stromal cells from human AGM region, fetal liver (FL) or bone marrow (BM) in Transwell non-contact system. According to the different culture methods, the EB cells were divided into 6 groups including EB control group, AGM group, FL group, BM group, AGM + FL group and AGM + BM group. The induced cells derived from EB were collected for Sca-1(+)/c-Kit(+) cells analysis by flow cytometry and colony forming unit (CFU) assay. The results showed that Sca-1(+)/c-Kit(+) cell proportion of EB cells significantly increased after being induced by different stromal cells (p < 0.01). The AGM + FL group had most Sca-1(+)/c-Kit(+) cells for the positive cell proportion reached (21.96 ± 2.54) % (p < 0.01). The Sca-1(+)/c-Kit(+) cell proportion of AGM + BM group was much high than that of BM group too (p < 0.01). The EB control group showed CFU amount less than any other groups, while the CFU amount of AGM + FL, AGM + BM groups were higher, especially in the AGM + FL group (p < 0.01). It is concluded that the human AGM region stromal cells may help to maintain certain number of primitive HSC with high proliferation potential. Human AGM region, FL or BM stromal cells, applied in sequential orders, can significantly expand in vitro the primitive hematopoietic stem/progenitor cells derived from ESC.

Sequentially inducting murine embryonic stem cells into hematopoietic stem cells in vitro by hematopoietic development procedure for reconstitution of hematopoiesis in vivo / 中国实验血液学杂志

This study was purposed to directly induce murine embryonic stem cells (ESC) into hematopoietic stem cells (HSC) by simulating the spatial and temporal hematopoietic microenvironment changes in embryonic development, and to investigate the function of in vivo hematopoietic reconstitution of these HSC. E14 ESC were induced into embryoid body (EB) firstly. Then the cells from EB were further co-cultured with human aorta-gonad-mesonephros (AGM) region, fetal liver (FL) and bone marrow (BM) stromal cells in Transwell non-contact system in sequential orders. After 6 days of each co-cultured stage, the induced cells derived from EB were collected to analyze the Sca-1(+)c-Kit(+) cells by flow cytometry, check teratoma formation and transplant to BALB/C female mice exposed to lethal dose (60)Co γ-ray. The recipient mice were divided randomly into 5 groups: AGM, AGM + FL, AGM + FL + BM, irradiation control and normal control groups. The survival rates, hematopoietic reconstitution and engraftment of donor cells in the different groups were monitored. The results showed that Sca-1(+)c-Kit(+) cell level in EB cells co-cultured with human AGM region and FL stromal cells reached to peak value (21.96 ± 2.54)%. Teratomas could be found in NOD-SCID mice after subcutaneous injection of EB cells co-cultured with human AGM region stromal cells, while there was no teratoma in the mice after subcutaneous injection of EB cells induced by human AGM region and FL stromal cells. The recipients in AGM group and irradiation control group all died. The survival rate was 77.8% in AGM+FL group, and 66.7% in AGM+FL+BM group. The peripheral blood cell count was near normal at day 21 after transplantation, and Sry gene copies from donor could be detected in recipient mice of these two groups. It is concluded that sequentially inductive system with feeder cells from human AGM region, fetal liver and bone marrow simulating embryonic defined hematopoiesis procedures can enhance the directed differentiation of ESC into HSC which can safely reconstitute hematopoiesis in vivo.

Isolation and culture of human embryonic AGM derived HSPCs in hematopoietic culture systems created by AGM stromal cells / 中国实验血液学杂志

This study was purposed to isolate human embryonic AGM derived HSPCs and investigate the effect of AGM stromal cells on AGM-derived HSPCs. Immunohistochemical sections of human AGM tissue were investigated for CD34, Flk-1 and VEGF expression. Human AGM-derived single cells were isolated and seeded onto pre-treated feeder of human AGM stromal cells (hAGMS3 and hAGMS4) by direct contact and non-contact co-culture in Transwell culture system. Growth characteristics of HSPCs with cobblestone area-forming cells (CAFCs) were observed and number of cobblestone area (CA) was counted. Indirect immunofluorescent assay was used to detect CD34 and Flk-1 expression on the surface of suspended cells as well as CAFCs in contact co-culture system. The cells after culture for 2 weeks were collected from both contact and non-contact co-culture systems for CFU assay. The result showed that hematopoietic cells in AGM tissue expressed CD34 and Flk-1. Both of the hematopoietic culture systems could produce CFCs. Nevertheless, direct contact co-culture produced CD34(+)Flk-1(+) CAFC and more CFUs than those from indirect non-contact culture (hAGMS3 system: 1647 +/- 194 vs 389 +/- 31, p < 0.05; hAGMS4 system: 1586 +/- 75 vs 432 +/- 35, p < 0.05). It is concluded that there were CD34(+)Flk-1(+) HSCs in human embryonic AGM region. The hematopoietic co-culture systems composed of AGM-derived HSPCs and AGM stromal cells are successfully established, both direct contact and Transwell non-contact co-culture can expand AGM-derived definitive HSPCs. Cell-cell contact between AGM-derived HSPCs and AGM stromal cells are of most importance to maintain and expand AGM-HSPCs.

BMP-4 and VEGF promote development of hematopoietic stem cells during the embryoid body formation from embryonic stem cells / 中国实验血液学杂志

The objective of this study was to explore the effects of BMP-4 and VEGF on the development of primary hematopoietic stem cells during the differentiation of embryonic stem cells (ESCs) into embryoid body (EB). Murine E14 ESCs were seeded into semisolid methylcellulose-based medium for EB formation. According to added or not cytokines, experiments were divided into: (1) group of spontaneous differentiation without cytokine as control; (2) group of BMP-4 in different concentrations (0, 5, 15, 25 and 50 ng/ml); (3) group of BMP-4 combined with VEGF; (4) group of VEGF alone. EBs were collected on days 3, 6, 9, 12, 15, and the proportion of Flk-1(+) cells were assayed by flow cytometry. The results showed that in the different BMP-4 concentration groups, the proportions of Flk-1(+) cells were significantly different, and it reached the peak values in 25 ng/ml BMP-4 group as 6.51 +/- 1.02% at day 3 and 7.70 +/- 1.12% at day 6 respectively, which were statistically higher than those in control group without-BMP-4 and in 5 ng/ml BMP-4 group (p < 0.05). When BMP-4 was used in combination with VEGF, Flk-1(+) cells went to peak proportion value at day 9 as 27.53 +/- 8.14%, which was statistically higher than that in spontaneous differentiation group as 8.77 +/- 2.35% (p < 0.05) and VEGF treatment group as 11.21 +/- 2.23% (p < 0.05). It is concluded that BMP-4 in combination with VEGF can promote Flk-1(+) cells genesis during EB formation in vitro, which provides experimental evidence for researches on directed differentiation of ESCs into hematopoietic stem cells simulating the microenvironment in vivo.

Expression of BMP-4 in stromal cells in vitro derived from human aorta-gonad-mesonephros region / 中国实验血液学杂志

The objective of this study was to investigate the expression of BMP-4 in stromal cells in vitro derived from human aorta-gonad-mesonephros (AGM) region. Stromal cells derived from human AGM region (hAGM S1-S5) and fibroblasts derived from human fetal trunk (hFT) were cultured in vitro. RT-PCR was used to analyze the expression of BMP-4 in hAGM S1-S5 stromal cells at mRNA level. And BMP-4 level was detected in the supernatant of hAGM S1-S5 stromal cells by ELISA assay. hFT cells were used as control group. The results showed that the heterogenous hAGM S1-S5 stromal cells displyed shapes of fibroblast-like and endothelial-like cells. hAGM S1-S5 stromal cells expressed BMP-4 mRNA, but fetal trunk fibroblasts (hFT) did not express BMP-4 mRNA. In the supernatant of hAGM S1-S5 cells, BMP-4 could be detected by ELISA assay ana its levels were statistically higher than that in hFT group (p < 0.05), while there was no significant difference between groups of hAGM S1-S5 (p > 0.05). It is concluded that human AGM-derived stromal cells in vitro express BMP-4, and the establishment of a new culture system based on the feeder cells of AGM stroma would promote the differention of embryonic stem cells into hematopoietic stem cells at a high proportion.

Hematopoietic growth factors expressed in human aorta-gonad-mesonephros (AGM)-derived stromal cells in vitro / 中国实验血液学杂志

This study was aimed to investigate the hematopoietic growth factors expressed in human aorta-gonad-mesonephros (AGM)-derived stromal cells in vitro in order to provide the basic data for elucidating the role of AGM -derived-stromal cells in embryo-hematopoiesis and its hematopoietic suppoitive effect. RT-PCR was used to analyze the expression of IL-6, SCF, Flt3-L, oncostatin M (OSM), IL-3, TPO, M-CSF and LIF in human aorta-gonad-mesonephros-derived stromal cells (hAGMS1-S5) at mRNA level. IL-6, SCF and Flt3-L levels were detected in the supernatant of hAGMS1-S5 stromal cells by ELISA assay. Umbilical cord blood CD34(+) cells were cocultured with hAGMS1-S5 feeder cells, and hematopoietic cells were collected at day 14 for colony analysis in methylcellulose semisolid medium. The results showed that human aorta-gonad-mesonephros-derived stromal cells S1-S5 expressed IL-6, SCF, Flt3-L and OSM mRNA, but did not express IL-3, TPO, M-CSF and LIF mRNA. In the supernatant of hAGMS1-S5 cells, IL-6, SCF and Flt3-L could be detected by ELISA assay at different levels, while there was no significant difference between groups of hAGMS1-S5 (P > 0.05). When cocultured with umbilical cord blood CD34(+) cells, hAGMS1-S5 could support the expansion of CFU-GM, BFU-E, and CFU-Mix. The supportive effects of hAGM S1-S5 were significantly different (P < 0.05), hAGM S3 and S4 were better than hAGM S1, S2, and S5. It is concluded that detection of hematopoietic growth factors expressed in human aorta-gonad-mesonephros-derived stromal cells provided a solid foundation to elucidate the mechanism of hematopoiesis and the hematopoietic supportive effect of these stromal cells.

Supportive effects of human aorta-gonad-mesonephros-derived stromal cells on umbilical cord blood LTC-IC / 中国实验血液学杂志

The objective of this study was to explore the supportive effects of human aorta-gonad-mesonephros (AGM)-derived stromal cells on human umbilical cord blood long-term culture-initiating cells (LTC-IC). A co-culture system was established with human AGM stromal cells and umbilical cord blood CD34(+) cells. Different stromal cells derived from human AGM region (hAGM S1-S5) were plated on 24-well plates as feeder cells. CD34(+) cells were positively selected from human umbilical cord blood through immunomagnetic bead selection method, seeded on the feeder cells, and co-cultured for 8 weeks. The hematopoietic cells were collected at 5, 6, 7 and 8 weeks for CFC analysis. Frequencies of LTC-IC in umbilical cord blood CD34(+) cells after co-culture with AGM stromal cells were detected through limiting dilute analysis (LDA). The results showed that there was no any hematopoietic CFC in the feeder cell-free culture system after 5 weeks of co-culture. However, in AGM feeder cells culture systems, there were still CFCs after 5 weeks of co-culture, which indicated that human AGM stromal cells could maintain LTC-IC in vitro. In groups of hAGM feeders, hAGMS3 and S4 had better supportive effects than other AGM groups (P < 0.05). The absolute number of LTC-IC in hAGM S3 and S4 culture systems got expansion up to (176 +/- 46)% and (187 +/- 52)% respectively without significant difference between hAGMS3 and S4 (P > 0.05). It is concluded that human AGM stromal cells S1-S5 support the maintenance of umbilical blood LTC-IC in vitro, while hAGMS3 and S4 cells have better effects on maintaining LTC-IC and expansion of LTC-IC.

Supportive effects of stromal cells from human embryonic aorta-gonad-mesonephros region on umbilical cord blood CD34+ cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the supportive effects of stromal cells from human aorta-gonad-mesonephros (AGM) region on umbilical cord blood CD34+ cells.</p><p><b>METHODS</b>Stromal cells derived from human AGM region (hAGMS1-S5) and fetal trunk fibroblasts (hFf) were cultured on the bottom of 24-well plates as feeder cells. CD34+ cells positively selected from human umbilical cord blood through immunomagnetic beads selection method, were seeded into 24-well plates, and co-cultured for 28d. The number of total nucleated cells (TNC), CD34+ cells, CD34+ CD38- cells, and CFC were counted every week.</p><p><b>RESULTS</b>Stromal cells from human AGM region significantly supported proliferation of the TNC, CD34+ cells, CD34+ CD38- cells and CFC, when compared with hFT and controls without feeder cells (P < 0.05). The TNC increased (25.13 +/- 4.83)-fold (peak value) at day 21 in group of co-culture with AGM stromal cells. CD34 and CD34+ CD38- cells increased (2.68 +/- 0.51)- and (2. 38 +/- 0.45)-fold respectively at day 14 of co-culture. In colony analysis, HPP-CFU increased (2.62 +/- 0.85)-fold at day 14 of co-culture. The supportive effects of human AGM S1-S5 were significantly different, hAGM S3 and S4 were better than hAGM SI, S2, and S5 (P < 0.05).</p><p><b>CONCLUSIONS</b>Human AGM stromal cells S1-S5 could support the maintenance and expansion of umbilical cord blood CD34+ cells in vitro. hAGMS3, S4 cell had better effects on maintaining HSC activity, which would provide model cells and basic data for researches on hematopoiesis mechanism and hematopoietic differentiation of embryonic stem cells.</p>

Changes of P-selectin and E-selectin in children with Kawasaki disease / 中华儿科杂志

<p><b>OBJECTIVE</b>Kawasaki disease (KD) is a kind of febrile disorder without definite etiology. The pathologic change of KD is characterized by nonspecific vasculitis, which mainly involves the coronary artery. Some patients may have coronary angioma formation, and some of them will result in the coronary narrowing or embolism. Notwithstanding that KD has been one of the most common causes for acquired heart diseases in childhood in addition to the rheumatic fever, the pathogenesis of the vascular damage remains unknown. This study was conducted to explore the pathophysiological role of cell adhesion molecules (P-selectin and E-selectin) on the endothelial lesions in KD, and to look for the evidence of direct relationship between the plasma levels of soluble cell adhesion molecules (P- and E-selectin) and the incidence of the coronary artery lesion (CAL).</p><p><b>METHODS</b>Soluble P-selectin (PS), E-selectin (ES), thromboxane-B(2)(TXB(2)), 6-keto-PGF(1)alpha (6-KPGF(1)alpha) were measured in 36 patients with KD, 20 patients with febrile disease and 30 healthy children by using double antibody sandwich enzyme linked immunosorbent assay (ELISA) and radioimmunoassay. Patients with KD were separated into acute phase group, subacute phase group, recovery phase group, coronary artery lesion group (CAL), non-coronary artery lesion group (NCAL), intravenous immunoglobulin (IVIG) effective group (body temperature back to normal after 48 hours of using IVIG), and IVIG ineffective group.</p><p><b>RESULTS</b>Plasma PS and ES levels in the acute phase group [(211 +/- 28 and 186 +/- 14) ng/ml], subacute phase group [(238 +/- 27 and 151 +/- 13) ng/ml] and recovery phase group [(198 +/- 21 and 1008 +/- 9) ng/ml] were significantly higher than those in the healthy group [(102 +/- 36 and 72 +/- 10) ng/ml, P < 0.01]. The plasma PS levels remained higher after the treatment, but in IVIG effective group, the PS and ES levels declined significantly (P < 0.01) compared with those in acute phase group. Plasma PS and ES levels of CAL group [(281 +/- 78 and 210 +/- 52) ng/ml] were significantly higher than those of NCAL group [(217 +/- 15 and 108 +/- 10) ng/ml, P < 0.01]. In contrast to 1 week after the treatment, the PS and ES in IVIG effective group at the time point of 2 weeks after the treatment decreased more significantly (P < 0.01). While the PS and ES in IVIG ineffective group remained higher at the time point of 2 weeks after the treatment, which showed no significant difference compared with those 1 week after the treatment (P > 0.05). One week after the treatment, the PS levels of IVIG effective and ineffective groups did not descend, and there was no significant difference in PS between these two groups at this time point. Two weeks after the treatment, the PS and ES in IVIG ineffective group remained higher than those in IVIG effective group, and there was a significant difference between them. The peak level of PS appeared in the subacute phase. TXB(2) levels of KD in acute phase group increased markedly, which were significantly higher than those of healthy group [(345 +/- 127 and 190 +/- 69) ng/L, P < 0.01]. There was no significant difference between subacute phase group and healthy group. No significant difference was found between CAL group and NCAL group (P > 0.05). The levels of TXB(2) declined quickly after the treatment. The 6-KPGF(1)alpha level in KD of acute phase group, subacute phase group and recovery phase group [(7.1 +/- 2.8, 10.8 +/- 3.7 and 11.3 +/- 4.0) ng/L, respectively] was significantly lower than that of healthy group [(17.7 +/- 5.8) ng/L, P < 0.01], and the levels did not recover to normal even 2 weeks after the treatment. There was no significant difference 6-KPGF(1)alpha levels between CAL group and NCAL group (P > 0.05). In the febrile group, PS and ES levels showed no significant differences compared with healthy children (P > 0.05). ES level of KD patients was significantly correlated with CRP levels (r = 0.79 P < 0.01). In febrile group, there was no significant correlation between ES and CRP. There was a significant correlation between PS and PLT levels in KD patients (r = 0.75 P < 0.01), and no significant correlation between PS and PLT levels in febrile patients.</p><p><b>CONCLUSION</b>The increase of plasma PS and ES levels in KD acute phase and subacute phase might play an important role in the pathophysiology of the endothelial damage. E- and P-selectin may potentially be a predictor of CAL in patients with KD.</p>