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.

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).

[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.

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.

[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.

[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).

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.

[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.

[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).

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.

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).

[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.

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.

[Effects of bone marrow endothelial cell conditioned medium on the expansion of mature megakaryocytes and CFU-Meg in vitro].

Objective: To investigate the effects of serum-free bone marrow endothelial cell conditioned medium (E-CM) and serum-free bone marrow fibroblast conditioned medium (F-CM) on the expansion of mature megakaryocytes and CFU-Meg .

Methods: E-CM and F-CM were collected and added in liquid culture system respectively.

Results: E-CM and F-CM significantly promoted the expansion of mature megakaryocytes and CFU-Meg in liquid culture system.

[Effects of bone marrow fibroblasts conditioned media on the expansion of megakaryocytes in vitro].

Objective: To investigate the effect of bone marrow fibroblasts conditioned media on the expansion of megakaryocytes in vitro.

Methods: Serum-free bone marrow fibroblasts conditioned medium (BMF-CM) was prepared and collected. The effect of BMF-CM or BMF-CM combined with IL-11 on the expansion of mature megakaryocytes and the effect of BMF-CM on the expansion of megakaryocytic progenitors were observed.

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.

[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.

[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.

[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 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.

[Analysis of the different effects of murine bone marrow endothelial cell conditioned medium on the growth of embryonic and adult hematopoietic stem/progenitor cells in vitro].

In the present study, the effects of murine bone marrow endothelial cell conditioned medium (mBMEC-CM) on the growth of yolk sac and bone marrow hematopoietic stem/progenitor cells (HSPC) were investigated. Nonadherent cells of yolk sac and bone marrow were collected for semisolid culture assay of CFU-GM and HPP-CFC after being cultured in DMEM with 10% FBS, 10% mBMEC-CM and/or FL (5 ng/ml), TPO (2 ng/ml) for 24 hours. The number of CFU-GM and HPP-CFC was counted by day 7 and 14 respectively.

Expression of hematopoietic inhibitory factors in mouse fibroblasts, macrophages and endothelial cells.

Pure bone marrow fibroblasts, macrophages and endothelial cells were cultured in Iscove-modified Dulbecco's medium. RT-PCR was used to determine the expression of inhibitory cytokine mRNAs in these cell types. Serum-free conditioned medium was collected from each cell type and ultrafiltration was performed with a centriprep 10.