Soluble factors from bone marrow endothelial cells regulate differentiation and proliferation of hematopoietic and endothelial lineages and embryonic stem cells.

We have established a bone marrow endothelial cell line. This review focuses on the elucidation and analysis of the effects of this bone marrow endothelial cell-conditioned medium (BMEC-CM) on the differentiation and proliferation of hematopoietic and endothelial progenitors as well as embryonic stem cells (ESCs). We will review that (1) BMEC-CM promotes proliferation and differentiation of hematopoietic lineage; (2) BMEC-CM promotes proliferation and differentiation of endothelial lineage; (3) BMEC-CM induces differentiation of hematopoietic stem cells/progenitors into endothelial progenitors; and (4) BMEC-CM induces differentiation of ESCs into hematopoietic cells and endothelial cells. We conclude that the soluble factors secreted by BMECs are able to support the proliferation and differentiation of hematopoietic and endothelium lineages. Moreover, these soluble factors induce hematopoietic cells to differentiate to endothelial cells, and induce ESCs to differentiate towards both endothelial cells and hematopoietic cells. Therefore, this work provides evidence that a close relationship involved in the development of hematopoietic and endothelial lineage. This disclosure will be beneficial for therapy strategy in the treatment of ischemic and tumor diseases, and improve our understanding of the relationship between hematopoietic and endothelial lineages.

An in vitro study of differentiation of hematopoietic cells to endothelial cells.

Bone-marrow-derived endothelial progenitor cells (BM-EPCs) contribute to postnatal neovascularization and therefore are of great interest for cell therapies to treat ischemic diseases. However, their origin and characteristics are still in controversy. In this paper, we identified the origin/lineage of the BM-EPCs that were isolated from bone marrow mononuclear cells and differentiated with the induction of bone-marrow endothelial-cellconditioned medium (ECCM). BM-EPCs were characterized in terms of phenotype, lineage potential, and their functional properties. Endothelial cell colonies derived from BM-EPC were cultured with ECCM for 3 months. Cultured EPC colony cells expressed endothelial cell markers and formed the capillary-like network in vitro. EPC colony cells expressed differential proliferative capacity; some of the colonies exhibited a high proliferative potential (HPP) capacity up to 20 population doublings. More importantly, these HPP-EPCs expressed hematopoietic marker CD45, exhibited endocytic activities, and preserved some of the myeloid cell activity. In addition, the HPP-EPCs secrete various growth factors including VEGF and GM-CSF into the culture medium. The results demonstrate that these EPCs were primarily derived from hematopoietic origin of early precursor cells and maintained high proliferative potential capacity, a feature with a significant potential in the application of cell therapy in ischemic diseases.

[Cultivation and purification of high proliferative potential endothelial progenitor cells from murine bone marrow].

The present study was designed to culture and purify high proliferative potential endothelial progenitor cells (HPP-EPCs) from murine bone marrow and their progeny cells using a culture system which has been established by us recently, in order for more intensive researches on these cells. Fresh murine bone marrow cells were preplated to allow the preferential attachment of non-EPCs to tissue culture plates and then unattached cells were repreplated in the culture system containing the bone marrow endothelial cell line-conditioned medium (BMEC-CM). The colonies containing about 2 x 10(4) cells were collected respectively and single colony-derived cells were cultured for their expansion in the above-mentioned culture system. These single colonies were able to yield 8 x 10(6) progeny cells. The endothelial-lineage characteristics of expanded cells were confirmed by immunofluorescent staining for CD31 and vWF, FITC-UEA-1 binding and Dil-Ac-LDL uptake. These data suggest that murine bone marrow HPP-EPCs can proliferate exuberantly so that their progeny cells can be obtained with high yield by using the single colony-derived cell culture in the presence of BMEC-CM.

GM-CSF accelerates proliferation of endothelial progenitor cells from murine bone marrow mononuclear cells in vitro.

OBJECTIVE: To test whether the GM-CSF accelerates the proliferation of bone marrow endothelial progenitor cells (BM EPCs). METHODS: BM EPCs were induced by endothelial cell conditioned medium (EC-CM). The effect of different concentrations of GM-CSF on the proliferation of BM EPCs was evaluated by the formation of EC-cols, MTT assay, and cell cycle assay. The single progenitor cell growth curves were quantified. RESULTS: The data indicated that GM-CSF accelerated the proliferation of BM EPCs both in colony numbers and colony size. MTT confirmed the effect of GM-CSF on accelerating the proliferation of BM EPCs. The single colony experiments showed that EC-cols expressed different proliferation capacity, suggesting that the EC-cols with different proliferation potentials might have been derived from different levels of immature progenitors. The cell cycle assay showed that the rate of cells entering into S phase was 9.3% in the group treated with GM-CSF and 2.1% in the controls. Furthermore, these cells displayed the specific endothelial cell markers and formed capillary-like structures. CONCLUSIONS: GM-CSF accelerates proliferation of BM EPCs. The potential beneficial of GM-CSF in the application of treating vascular ischemic patients is promising.

[Bone marrow endothelial cell-conditional medium promotes the generation of hematopoietic colony-forming cells from murine embryonic stem cells].

OBJECTIVE: To observe the inductive efficiency of deriving hematopoietic colony-forming cells from murine embryonic stem (mES) cells co-cultured with bone marrow stromal cell-conditional medium (mBMEC-CM). METHODS: After the day-4 embryoid bodies (4 dEBs) were derived from embryonic stem cell-D3 (ES-D3) cells, the cells of 4 dEBs were induced into hematopoietic colony-forming cells by co-culturing with mBMEC-CM. The numbers of 4 dEB-derived hematopoietic colonies (high proliferation potential-colony formation cells and burst forming unit-erythroid, HPP-CFC and BFU-E) were detected to explore the relation between the implanted 4 dEB-derived cell numbers and the colony numbers of BFU-E and HPP-CFC. The inducing effect of mBMEC-CM was observed according to the doses and days of induction. RESULTS: The number of 4 dEB-derived cells within 1 x 10(7)-4 x 10(7)/L was positively related to the colony numbers of HPP-CFC and BFU-E (HPP-CFC, r=0.916,P< 0.05; BFU-E, r=0.927, P<0.05). The inducing doses of mBMEC-CM within 0-20% were positively related to the colony numbers of HPP-CFC and BFU-E (HPP-CFC, r=0.909, P<0.05; BFU-E, r=0.927, P<0.01). The colony numbers of HPP-CFC and BFU-E derived from the 4 dEB-derived cells were the highest after 3 days of induction, followed by those of 6 days and 9 days. CONCLUSION: Bone marrow endothelial cell-conditional medium can promote the generation of HPP-CFC and BFU-E from murine embryonic stem cells.

Purification and growth of endothelial progenitor cells from murine bone marrow mononuclear cells.

This study reports the culture and purification of murine bone marrow endothelial progenitor cells (EPCs) using endothelial cell-conditioned medium (EC-CM). Endothelial-like cells appeared at day 5 in culture of bone marrow mononuclear cells in the presence of EC-CM in the culture system, and these cells incorporated acetylated low-density lipoproteins (Ac-LDL) and reacted with endothelial-specific Ulex Europaeus Lectin. Continued incubation of these cells at low density with EC-CM for longer than 10 days resulted in the formation of endothelial cell colonies which gave rise to colonies of endothelial progeny and can be passed for many generations in the EC-CM culture system. Cells derived from these colonies expressed endothelial cell markers such as vWF and CD31, incorporated Dil-Ac-LDL, stained positive for Ulex Europaeus Lectin, formed capillary-like structures on Matrigel, and demonstrated a high proliferative capacity in culture. These bone marrow-derived adherent cells were identified as EPCs. The purification and the formation of EPC colonies by using EC-CM were associated with the cytokines secreted in the EC-CM. VEGF, bFGF, and GM-CSF in the EC-CM stimulated the proliferation and growth of EPCs, whereas AcSDKP (tetrapeptide NAc-Ser-Asp-Lys-Pro) in EC-CM suppressed the growth of mesenchymal stem cells (MSC) and fibroblasts. This approach is efficient for isolation/purification and outgrowth of bone marrow EPCs in vitro, a very important cell source in angiogenic therapies and regenerative medicine.

[Long-term subculture and biological characterization of the murine bone marrow endothelial cell line].

The murine bone marrow endothelial cell line (mBMEC) has been maintained by means of subculture and cryopreservation for over 10 years since it was established in our laboratory. This study was aimed to newly identify biological characteristics of this cell line for further study. The cultured mBMEC cells were observed by inverted microscopy and transmission electron microscopy (TEM). PECAM-1 (CD31) and von Willebrand factor (vWF) were detected by immunofluorescent staining. The phagocytotic activity of the cells in culture was tested by using fluorescent acetylated low-density lipoprotein (Dil-Ac-LDL). The cell growth kinetics analysis and karyotype analysis were performed. The results showed that the adherent cells were mostly elliptical, rounded and spindle-shaped, and some of them connected to each other to form cord- and network-like arrangements in mBMEC cultures at subconfluence. The adherent cells grew up to confluence as a cobblestone-like monolayer. Several ultrastructural features of the endothelial cells could be observed in TEM sections of the cultured cells. More than 94% of mBMEC cells were positive for either CD31 or vWF. The phagocytotic ingestion of Dil-Ac-LDL occurred in 98.5% of cells. In normal culture conditions, the cells grew with a mean population doubling time of 54.6 hours and the maximal mitotic index was 38 per thousand in the rapid growth period. The colony yields were 4.33% to 7.40% depending on the plating density of cells. Karyotypes of all the cells were aneuploidy with a greater percentage of hyperdiploid. It is concluded that mBMEC cells retain the fundamental properties of endothelial cells, but the growth kinetics and biological behaviors are slightly different from those in the early days after the establishment of this cell line.

Inducing effects of macrophage stimulating protein on the expansion of early hematopoietic progenitor cells in liquid culture.

BACKGROUND: Macrophage stimulating protein (MSP) is produced by human bone marrow endothelial cells. In this study, we sought to observe its effects on inducing the expansion of early hematopoietic progenitor cells which were cultured in a liquid culture system in the presence of the combination of stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), granulocyte macrophage-colony stimulating factor (GM-CSF), erythropoietin (EPO) (Cys) and MSP or of Cys and bone marrow endothelial cell conditioned medium (EC-CM). METHODS: Human bone marrow CD34(+) cells were separated and cultured in a liquid culture system for 6 days. Granulocyte-macrophage colony forming unit (CFU-GM) and colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-GEMM) were employed to assay the effects of different treatment on the proliferation of hematopoeitic stem/progenitor cells. The nitroblue tetrazolium (NBT) reductive test and hoechest 33258 staining were employed to reflect the differentiation and apoptosis of the cells respectively. RESULTS: MSP inhibited the proliferation of CFU-GM and CFU-GEMM in semi-solid culture and the inhibitory effect on CFU-GEMM was stronger than on CFU-GM. MSP inhibited the differentiation of early hematopoietic progenitor cells induced by hematopoietic stimulators. Bone marrow (BM) CFU-GEMM was 2.3-fold or 1.7-fold increase or significantly decreased in either Cys + EC-CM, Cys + MSP or Cys compared with 0 hour control in liquid culture system after 6 days. CONCLUSION: MSP, a hematopoietic inhibitor, inhibits the differentiation of early hematopoietic progenitor cells induced by hematopoietic stimulators and makes the early hematopoietic progenitor cells expand in a liquid culture system.

[Effects of granulocyte-macrophage colony stimulating factor on growth of murine bone marrow endothelial cells].

The purpose of this study was to investigate the effects of granulocyte-macrophage colony stimulating factor (GM-CSF) on the growth of mouse bone marrow endothelial cells. Endothelial cell culture medium (Endo-M) was used to culture murine bone marrow endothelial cells. Endothelial cell colonies were counted under microscope by Wright-Giemsa staining. The effect of different concentration of GM-CSF on the proliferation of bone marrow endothelial cells was observed by the formation of endothelial cell colonies, MTT and flow cytometry. The results indicated that the endothelial specific marker vWF was expressed by the colony cells, GM-CSF promoted the proliferation of bone marrow endothelial cell colonies and MTT confirmed the effect of GM-CSF on promoting the proliferation of bone marrow endothelial cells. The result of detecting cell cycle showed that the rate of cells entering into S phase was 9.3% in GM-CSF added group and the rate of cells entering into S phase was 2.1% in control. There was no significant difference in cell growth curve between the first passage and fourth passage. It is concluded that GM-CSF can promote the proliferation of bone marrow endothelial cells, the proliferation potential of bone marrow endothelial cells between the first and fourth passage no significantly changes.

[Effects of endothelial cells from cord blood on the amplification of human early hematopoietic cells from cord blood in vitro].

OBJECTIVE: To observe the effects of endothelial cells from umbilical cord blood (UCB) on the amplification of human early hematopoietic cells from UCB in vitro. METHODS: Endothelial cells from UCB were cultured by the optimized medium of endothelial cells. There were 2 experiment groups: cytokines group (SCF+IL-3+IL-6+GM-CSF, CKs group) and noncontact group (endothelial cell layer with CKs without contacting the CD34+ cells group). CD34+ cells from UCB were isolated by MiniMACS. After the cells in the CKs group and the noncontact group were cultured for 7 days, the amplifying folds of early hematopoietic cells were assayed. RESULTS: Early hematopoietic cells from UCB were expanded in the CKs group or the noncontact group. The amplifying folds of the noncontact group on early hematopoietic cells were significantly more than those of the CKs group. CONCLUSION: The amplification effect of the noncontact group on early hematopoietic cells is superior to that of the CKs group.

Two conditional media promoting the differentiation murine embryonic stem cells into hematopoietic stem cells.

OBJECTIVE: To derive hematopoietic stem cells with functional properties of hematopoietic reconstitution from murine embryonic stem (ES) cells. METHODS: ES-D3 cells by formation of the day-4 embryoid bodies (4dEBs) were induced into hematopoietic stem cells by co-culture with murine bone marrow endothelial cell-conditional medium (mBMEC-CM) and the fetal liver stromal cell-conditional medium (FLSC-CM). This experiment was designed to 4 groups (mBMEC-CM + FLSC-CM group, mBMEC-CM group, FLSC-CM group, and the control group). RESULTS: The total cell numbers, CD34+ cell numbers, and colony numbers formed in the mBMEC-CM+FLSC-CM group were the highest among the 4 groups. The cells in the mBMEC-CM + FLSC-CM group resumed the hematopoietic system of the mice after being transplanted with the inducing cells. CONCLUSION: The culture condition combing mBMEC-CM with FLSC-CM can promote murine ES cells differentiating into hematopoietic stem cells with functional properties of hematopoietic reconstitution.

Thymosin beta4 and AcSDKP inhibit the proliferation of HL-60 cells and induce their differentiation and apoptosis.

Our previous works have shown that bone marrow stromal cells secrete thymosin beta4 (Tbeta4) and AcSDKP. Tbeta4 and AcSDKP are existed in the conditioned medium of bone marrow endothelial cells. They exerted inhibitory effects on hematopoietic cells and then had protective effect on the early hematopoietic cells, which were cultured in the presence of hematopoietic stimulators. Thymosin beta4 consists of 43 peptides with a molecular weight of 4963. It contains at its N-terminal end the sequence of the acetylated tetrapeptide Ac-N-Ser-Asp-Lys-Pro (AcSDKP). This study was performed to evaluate the effect of Tbeta4 and AcSDKP on the growth of HL-60 cells. It was showed that Tbeta4 (10(-11)-10(-7)mol/L) and AcSDKP (10(-11)-10(-7)mol/L) had the dose-dependent inhibitory effect on the proliferation of HL-60 cells. Based on cell morphology and NBT reduction, Tbeta4 and AcSDKP induced differentiation of HL-60 cells. Morphologic and DNA fragment analysis proved that Tbeta4 and AcSDKP induced apoptosis of HL-60 cells. In order to analyze the mechanism of the effects of Tbeta4 and AcSDKP, intracellular free Ca(2+) concentration ([Ca(2+)](i)) of HL-60 leukemic cells was tested and Atlas cDNA Expression Array was performed. The results showed that Tbeta4 and AcSDKP could increased [Ca(2+)](i) by stimulating the release of Ca(2+) from intracellular Ca(2+) pool. Moreover, AcSDKP could also elicit a potent extracelluar calcium influx in HL-60 cells. Tbeta4 could also change apoptotic-related gene expression in leukemic cells, and resulted in the inhibition of proliferation and induction of differentiation and apoptosis of leukemic cells.

[Effect of hematopoietic stimulating factors on the expansion of megakaryocyte].

OBJECTIVE: To investigate the effect of hematopoietic stimulating factors on the expansion of mature megakaryocytes. METHODS: (2, 4, 6, 8, 10) x 10(5)/mL bone marrow single nucleus cells (BMNC) were added in the culture system of colony forming unit-megkaryocyte (CFU-Meg) to find out the relationship of the cultured BMNC with the output of CFU-Meg. rmSCF + rmTPO + rmIL-3 (3HSFs) and rmSCF + rmTPO + rmIL-3 + rmIL-6 (4HSFs) or F-CM were added in the liquid culture system of megkaryocytes respectively. The number of mature megakaryocytes were counted every other day. RESULTS: The number of CFU-Meg increased with the increase of the cultured BMNC. The CFU-Meg productivity of 1 x 10(6) BMNC/mL culture system was more than that of 2 x 10(5) BMNC/mL culture system. 3HSFs and 4HSFs or F-CM significantly promoted the expansion of mature megakaryocytes in the liquid culture system, but the effect was different. The peak time of the number of mature megakaryocytes in 3HSFs and 4HSFs or F-CM were 7 d, 7 d and 5 d respectively. CONCLUSION: 3HSFs and 4 HSFs or F-CM had positive effect on the expansion of mature megakaryocytes. 4HSFs was better than 3HSFs and F-CM. 3HSFs was better than F-CM. The peak time of the number of mature megakaryocytes in different culture systems was different.

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.

[Differentiation from human embryonic stem cells to hematopoietic cells and endothelial cells].

Embryonic stem cells are pluripotent and their differentiation in vitro can serve as an experimental model to explore the molecular mechanisms of early embryonic development. To investigate the effect of stromal cell conditioned medium combined with cytokines (sccm + cys) on the differentiation from human embryonic stem cells to hematopoietic cells and endothelial cells, the mouse fibroblast feeder cells to make human embryonic stem cells grown into embryonic bodies (EBs) were initially deleted. After culture for 3 days, EB cells were trypsinized into single cells and induced for 8 days by sccm + cys. Then, the differentiated cells were cultured in the semisolid medium containing 0.9% methylcellulose and cytokines to study the colony forming and self-renewal ability of cells. Immunocytochemical staining was used to check the surface markers of the colony cells. During the induction, mRNA expression of flk-1, BMI-1, scl, and Zeta-globin genes was tested by RT-PCR. Surface markers, such as flk-1, CD34 were tested by the flow cytometry. The results demonstrated that: (1) cell clusters containing 20-30 cells were formed after culture for 8 - 14 days in the semisolid medium, replanting these cells resulted in similar cell cluster forming. In addition, CD45 positive in big cell colonies were also found in the semisolid medium; (2) attached cell colonies appeared after culture for 8 days in the semisolid medium and VIII factor, UEA and KDR could be detected as negative by immunocytochemical staining; (3) on the 4(th) day of induction, mRNAs of flk-1, BMI-1, scl and Zeta-globin were all expressed. On the 8(th) day of induction, all of the above genes except Zeta-globin were expressed, while ES cell and EB cells which served as controls did not express scl and Zeta-globin genes; (4) on the 8(th) day of induction, the proportions of flk-1(+) cells and CD34(+) cells among all the inducing population were 9.8% and 16.8%, respectively, while the corresponding positive populations were 0.36% and 1.16% in spontaneously differentiated 11(th) day's EB, and 0.04% and 0.16%, respectively, in ES cells. If is concluded that embryonic stem cells can differentiate into hematopoietic cells and endothelial cells in combinant culture system of this study.

[Promoting effects of serum-free murine bone marrow endothelial cell conditioned medium on the growth of bone marrow endothelial cells].

To study the effects of serum-free murine bone marrow endothelial cell conditioned medium (mBMEC-CM) on the growth of bone marrow endothelial cells, mBMEC-CM was collected and ultrafiltrated by Centriprep-10. The retentate of mBMEC-CM [molecular weight (MW)>10 kDa] and the filtrate of mBMEC-CM (MW<10 kDa) were obtained. The effect of bone marrow conditioned media, their components and exogenous cytokines on the formation of endothelial cell colonies were observed. The effect of bone marrow conditioned media, their components and exogenous cytokines on the proliferation of murine bone marrow endothelial cells were determined by [(3)H]-thymidine incorporation. The method of hybridizing to the Atlas cDNA array was used to determine the expression of cytokine mRNAs in bone marrow endothelial cells. The results obtained are as follows: vWF was expressed in bone marrow endothelial cells. The original mBMEC-CM and MW>10 kDa component of mBMEC-CM promoted the proliferation of bone marrow endothelial cell colonies and increased [(3)H]-thymidine incorporation of bone marrow endothelial cells. The MW<10 kDa component did not affect the production of endothelial cell colonies and did not increase [(3)H]-thymidine incorporation of endothelial cells. Six cytokines (IL-6, IL-11, SCF, GM-CSF, VEGF, bFGF) promoted the proliferation of bone marrow endothelial cell colonies. VEGF, bFGF and SCF increased [(3)H]-thymidine incorporation of bone marrow endothelial cells. According to the results of the Atlas cDNA array, GM-CSF,TGF-beta,BMP-2, bFGF, SCF, endothelin-2, thymosin beta10, MSP-1, connective tissue GF, PDGF-A chain, MIP-2 alpha, PlGF, neutrophil activating protein ENA-78, INF-gamma, IL-1, IL-6, IL-13, IL-11, inhibin-alpha mRNAs were expressed in endothelial cells. These results suggest that murine bone marrow endothelial cell conditioned medium promotes the proliferation of bone marrow endothelial cells.

[Stimulation of bone marrow stromal cells conditioned medium on the expansion of mature megakaryocytes and colony forming unit-megakaryocyte in vitro].

In this study the effects of bone marrow stromal cells conditioned medium on the expansion of mature megakaryocytes and colony forming unit-megakaryocyte (CFU-Meg) in vitro were investigated. The serum-free bone marrow fibroblast conditioned medium (F-CM), bone marrow endothelial cell conditioned medium (E-CM) and bone marrow macrophage conditioned medium (M-CM) were collected and ultrafiltrated by using Centriprep-10. F-CM, E-CM, M-CM, the retentate (>10 kDa F-CM, >10 kDa E-CM and >10 kDa M-CM) contained substances whose molecular weight was more than 10 kDa and the filtrate (<10 kDa F-CM, <10 kDa E-CM and <10 kDa M-CM) contained substances whose molecular weight was less than 10 kDa were added in liquid culture system respectively. The results showed that F-CM, >10 kDa F-CM and E-CM, >10 kDa E-CM significantly promoted the expansion of mature megakaryocytes in liquid culture system, the percentage of mature megakaryocytes compared with 0 h control were (287.33-/+16.77)%, (236.67-/+39.72)%, (141.21-/+17.47)% and (179.03-/+30.98)%. But <10 kDa F-CM, <10 kDa E-CM, M-CM, >10 kDa M-CM and <10 kDa M-CM had no positive effects on the expansion of mature megakaryocytes. The effects of F-CM, E-CM or M-CM on the expansion of CFU-Meg were also investigated. The results indicated that F-CM and E-CM promoted the expansion of CFU-Meg in liquid culture system. M-CM had no positive effect on the expansion of CFU-Meg. the percentage of CFU-Meg compared with 0 h control were (168.18-/+30.24)%, (215.17-/+17.4)% and (85.0-/+7.0)%. The results of reverse transcription-polymerase chain reaction (RT-PCR ) showed that transforming growth factor -beta1 (TGF-beta1) mRNA was expressed in bone marrow endothelial cells and was not expressed in bone marrow fibroblasts. Thrombopoietin (TPO) mRNA was expressed in bone marrow fibroblasts and was not expressed in bone marrow endothelial cells. These results suggest that F-CM, >10kDa F-CM and E-CM, >10kDa E-CM significantly promoted the expansion of mature megakaryocytes and CFU-Meg in liquid culture system. The effect of F-CM on the expansion of mature megakaryocytes is better than that of E-CM.

The effects and the mechanism of YSEC-CM on the growth of yolk sac hematopoietic stem/progenitor cells.

To explore the distinct background of bone marrow and embryonic yolk sac hematopoiesis performance, serum free murine yolk sac endothelial cell and bone marrow endothelial cell conditioned medium were compared for their effects on the development profiles of yolk sac hematopoietic stem/progenitor cells. The mRNAs expression techniques were applied to understand the cytokine and receptor genes expression and the possible mechanisms. The results suggested that the differential gene expressions were existed between the hematopoietic cells of yolk sac and bone marrow and between the microenvironment of yolk sac and bone marrow.