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Objective: To explore the role of bone marrow niche in the chemotherapy resistance of patient with acute myeloid leukemia (AML), and to investigate the effects of the MSCs on the apoptosis of HL-60 cell and its underlying mechanisms., Methods: MSCs were derived from the bone marrow of newly diagnosed AML patients (AML-MSCs) and health donors(MSCs) were co-cultured with HL-60 cells respectively. The apoptosis of HL-60 cells in the presence/absence of MSCs and/or Daunorubicin were determined by flow cytometry with Annexin V/PI double staining. In addition, the morphological features of HL-60 cells were observed by Wright-Giemsa staining, and the ratio of blasts and differentiated cells were counted. Furthermore, the expressions of apoptosis-related factors including Caspase-3, Caspase-8,Caspase-9 and Survivin were detected by Western blot., Results: The flow cytometry showed that there was no significant change in apoptosis of HL-60 cells co-cultured with MSC derived from healthy donors or AML patients. After adding Daunorubicin into different cultural systems, the apoptotic rates of HL-60, HL-60 co-cultured with normal MSCs and HL-60 co-cultured with AML-MSCs were (49.57±7.44)%, (30.72±4.05)% and (22.99±4.08)%, respectively, which showed that normal MSCs and AML-MSCs could remarkably supress Daunorubicin-induced HL-60 apoptosis, however, there was no statistically significant difference of apoptosis between HL-60 co-cultured with normal MSCs and HL-60 co-cultured with AML-MSCs. Wright-Giemsa staining showed that most of the HL-60 cells co-cultured with AML-MSCs were primitive, and cell differentiation was unusual. In AML-MSCs co-cultured group, the cell apoptosis and differentiation caused by DNR was significant decreased, and most of HL-60 cells were initial. Western blot showed that the cleavage activity of Caspase-3 of HL-60 in AML-MSCs and normal MSCs co-cultured group was decreased, compared with HL-60 in single cultured group, moreover, the decrease was significantly in AML-MSC group. Additionally, the expression of survivin in AML-MSCs and normal MSCs co-cultured group was increased, compared with that in single cultured group, and increase was significant in AML-MSCs group., Conclusion: MSCs can suppress Daunorubicin-induced HL-60 apoptosis via inhibiting Caspase-3 and maintaining survivin level.

Objective: To explore the role of bone marrow microenvironment(niche) in the development of acute myeloid leukemia (AML) and the effect of AML patients-derived MSC on the proliferation, cell cycle and immuno-phenotypes of HL-60 cells., Methods: The MSC derived from bone marrow of patients with newly diagnosed AML were isolated and co-cultured with HL-60 cells. The effect of MSC on proliferation of HL-60 cells was detected by using 3H-TdR incorporation method, the cell cycle and immunophenotypes of HL-60 cells were detected by flow cytometry., Results: The results of 3H-TdR incorporation assay showed that both AML-MSCs and normal MSCs remarkably suppressed the HL-60 cell proliferation in a time- and dose-dependent manner. The results of cell cycle analysis demonstrated that AML MSCs and normal MSCs induced arrest of the HL-60 cells in G 0 /G 1 phase. The results of immunophenotyping revealed that MSCs suppressed the expression of CD11a and CD154 on the surface of HL-60 cells. Moreover, AML MSCs exhibited increased inhibitory effects than that of normal MSCs. However, no remarkable effect of MSCs on CD54 expressions of HL-60 cells was observed in the current study., Conclusion: AML-MSCs possess effects on HL-60 cell proliferation, cell cycle and immunophenotypes similiar to normal MSCs, but exhibited increased suppressive capacity on the expression of CD11a and CD154.

Objective: To study the influence of acute myeloid leukemia (AML) microenvironment on mesenchymal stem cells (MSCs)., Methods: MSCs were isolated from the bone marrow of newly diagnosed AML patients (AML-MSCs) and were cultured. The morphology of MSC was observed by inverted microscopy, the immunophenotypes of MSC were detected by flow cytometry, the proliferation ability of MSC was detected by using MTT method, the multi-differentation ability of MSC was assayed by osteogenic, lipogenic and chrondrogenic induction. The morphologic features, immunophenotypic characteristics, cell proliferation, and multipotential differentiation capability were compared between the MSC derived from normal healthy donors and AML patients., Results: AML-MSCs presented the morphological features similar to the normal MSCs. In addition, AML-MSCs highly expressed CD29, CD44, CD73, CD105 and HLA-ABC. Meanwhile, they were homogenously negative for CD14，CD31, CD34, CD45, CD80, CD86 and HLA-DR. Further-more, AML-MSCs showed cell proliferation ability similar to normal MSCs. Notably, AML-MSCs exerted increased osteogenic-differentiation capacity as compared with normal MSCs., Conclusion: AML-MSCs possess typical MSC phenotypes but displayed enhanced osteogenic-differentiation capacity.

Objective: To aimed at the establishment of mouse stably knockout of MYSM1 mesenchymal stem cell(MSC) line C3H10T1/2, and to investigate its immunological capacity of MSC in vitro., Methods: To establish the stably transfected MSC cell line by using CRISPR-Cas9 technology. Then the Flow cytometry, quantitative PCR and Western blot were employed to detect whether the MYSM1 have been knockout yet. Furthermore, the immune modulatory effect of MYSM1 -/- MSC was tested by addition of MYSM1 -/- MSC supernatant into spleen lymphocyte and Foxp3 culture. The mRNA expression of inflammatory cytokines such as interleukin-4, interferon-γ and interleukin-17 were detected by quatitatine PCR., Results: The expression of MYSM1 was steadily knock out in MSC. In addition, MYSM1 -/- MSC showed a stronger inhibitory effect on the expression of inflammatory cytokines. Therefore, the MYSM1 has been stably knocked out in C3H10T1/2., Conclusion: The mouse stably knockout of MYSM1 mesenchymal stem cells has been successfully established, the knock-out of MYSM1 in MSC can induce more potent immunosuppressive effects on cellular immune reaction in vitro. Our data laid a foundation for the further MSC-based applications in immune related diseases.

Objective: To explore the effects of the shock wave on the capacity of mesenchymal stem cells(MSCs) to proliferate and differentiate into osteoblasts., Methods: MSCs were isolated from the bone marrow of healthy donors. The human bone marrow MSCs(BM-MSCs) were divided into 3 groups including blank control group,osteoinduced group and shock wave group. The MSCs in blank control group were cultured with common mediam; the MSCs in osteoinduced group were treated with osteogenic agents and cultured; the MSCs in shock wave group were cultured with common medium and stimulated by shock wave. The morphology of MSCs in each groups were observed by micoscopy; the CCK-8 was used to detect the proliferation ability of MSCs; the alkaline phosphatase staining and von Kossa staining were used to evaluale the differentiation potential of MSCs in each groups., Results: The results of CCK-8 revealed the shock wave could promote cell proliferation as compared with blank control group. The results of alkaline phosphatase and Von Kossa staining showed that the shock wave displayed a stronger ability to promote the human BMMSC differentiation into osteoblasts cells in comparison with the osteoinduced group. The blank control group was weakly positively stainined., Conclusion: The shock wave treatment can promote proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.

Objective: To isolate platelet-rich plasma(PRP) from the white slurry(WS), a depleted fraction of the clinical blood supply, so as to provide an easier method to harvest PRP for related studies and clinical use., Methods: The protocols preparing PRP from whole blood and WS were compared. The morphological characteristics of the different PRPs were observed under transmission electron microscope; the expression of the platelet markers CD41a and CD42b were detected by the flow cytometry. Moreover, the ingredients of the PRPs were measured by using cytoanalyzer. for detecting the physiological function of the PRP, the harvested PRP were added to MSC culture and the cell proliferation was detected by using CCK-8 method., Results: a large amount of PRP from WS was easier harvested. the WS-derived PRP shared similar morphological characteristics and ingredients as compared with whole blood-derived PRP. Importantly, the WS-derived PRP exhibited a higher expression of CD41a and CD42b than that of traditional PRP, which indicate that the WS is a promising reservoir for PRP., Conclusion: The WS can be used to prepare PRP, and the novel PRP share similar biological characteristics as traditional PRP prepared from whole blood. The present study provides an easier and economical method to harvest PRP and this findings may be helpful for PRP related studies.

Objective: To investigate the modulatory effect of the MSC derived from low attaching culture systems (suspending MSC) on T lymphocytes and the related mechanisms., Methods: The suspending MSC were generated from mouse compact bones by using low attaching plates and adherent cell culture flasks, respectively. The morphology of suspending MSC was observed under the inverted microscope and the cells were induced to differentiate into osteoblasts and adipocytes. Further, the surface antigen profile of MSC was analyzed with flow cytometry. In addition, the culture medium (CM) of suspending MSC and adherent MSC was collected and added into the activated T cell cultures before detection of the proliferation by CFSE assay. Moreover, the modulaory effects of the CM on the T cell-derived cytokines were detected by quantitative PCR. Also, the mRNA expression of cytokines of MSC was detected., Results: The suspending MSC grew in floating cell spheres and differentiated into osteoblasts and adipocytes in the induction medium. Furthermore, the suspending MSC shared the typical immuno-phenotype with their adherent counterparts. In addition, the results of CFSE assay demonstrated that suspending MSC derived CM suppressed ConA induced T cell proliferation. The results of quantitative PCR revealed that suspending MSC expressed transforming factor β1 and interleukin-6 at a higher level and suppressed the T cell expressing interferon γ and interleukine-17A., Conclusion: The suspending MSC exerted an unique modulatoy effect on T cells, which is quite different to adherent MSC.

Mesenchymal stem cells (MSC) have the capacities of low immunogenicity, multiple differentiation, hematopoietic supporting and immunoregulation. And due to their relative ease of availability and ex vivo expansion, the applications of MSC in the prevention and treatment of clinical disease have been rapidly expanded in the recent years. However, increasing investigations indicate that intravenously infused MSC widely distribute to various organs of the recipients. The two intended clinical goals of adoptive cellular therapy reached to the greatest efficiency. Therefore, the ideal candidate cells showed to have the capacity of site-specific relocation in vivo. In this review, the distribution characteristics of infused MSC and the recent research advances on the strategies to enhance targeted migration of MSCs are summarized.

This study was purposed to establish a convenient and efficient method for isolating and culturing mouse bone marrow mesenchymal stem cells (MSC). The femurs and tibias of mouse were taken under sterile condition. MSC were isolated and cultured with flushing- out bone marrow or collagenase-digested bone fragment or bone marrow plus bone fragment. MSC colony number and size were compared. Immunophenotype and differentiation ability were tested to identify MSC. The results showed that colonies from bone marrow plus bone fragment group came out earliest and the colony number was 20 ± 4 at day 4; there were 11.5 ± 2.5 colonies in collagenase-digested bone fragment group and 9.5 ± 1.5 in flushing- out bone marrow group. The total cell yields of MSC after passaging showed best in bone marrow plus bone fragment group. Flow cytometry data showed the cultured cells expressed Sca-1, CD44 and CD29, not expressed pan-leukocyte surface marker CD45 and endothelial cell marker CD31. The isolated and cultured MSC could differentiate into osteoblast at the osteogenic differentiation condition, or adipocyte at adipogenic differentiation condition. It is concluded that the method of bone marrow plus bone fragment is convenient and efficient for isolating and culturing MSC.

This study was purposed to investigate the regulatory effects of differentiating mesenchymal stem cells (MSC) on osteoclast formation. The MSC from mouse compact bones were cultured and induced into osteoblasts and adipocytes for one week. To test their regulatory effect on osteoclastogenesis, osteogenically differentiated and adipogenically differentiated MSC were co-cultured with CD11b(+) monocytes and osteoclasts were identified with in situ tartrate-resistant acid phosphatase (TRAP) staining. The results showed that differentiated MSC supported osteoclastogenesis but the osteoclast supporting capacity of osteogenically differentiated MSC decreased as compared with undifferentiated MSC. More interestingly, the adipogenically differentiated MSC significantly promoted osteoclasts formation when co-cultured with monocytes. It is concluded that the regulatory effect of MSC on osteoclast formation has changed while they have differentiated into different types of cells. The findings indicate that MSC may exert alternative effect on osteoclastogenesis by differentiation to descendant cells.

This study was purposed to investigate the effect of RUNX1 on transcription activity of WNT5A promoter in mouse bone marrow derived mesenchymal stem cells (MSC), and to explore the mechanism by which bone marrow environments regulate MSC. RT-PCR was used to detect the expression of RUNX1 in MSC isolated from mouse bone marrow and cultured in vitro; the chromatin immunoprecipitation (ChIP) was used to investigate the direct in vivo interaction between the RUNX1 and WNT5A promoter; retrovirus system was utilized to introduce the RUNX1 gene into MSC to detect the regulation of RUNX1 on the transcription activity of WNT5A promoter. The results showed that mouse bone marrow derived MSC was positive for Oil Red O, van Kossa and toluidine blue staining respectively and RUNX1 expressed in MSC. WNT5A promoter could be bound by RUNX1, and the expression level of WNT5A was enhanced with the increase of RUNX1. It is concluded that RUNX1 expresses in mouse bone marrow derived MSC, WNT5A is a direct target gene of RUNX1 and its transcriptional activity is regulated by RUNX1.

This study was aimed to investigate whether endothelium-specific deletion of PTEN can affect hemangioblast development in the AGM region of mouse embryos. Based on Cre/loxP system, the Tie2CrePten(loxp/loxp) and Tie2CrePten(loxp/wt) mouse embryos were obtained. The genotype was identified by PCR. After treated with type I collagenase, the AGM region was dispersed into single-cell suspension, and then was cultured in blast colony-forming cell (BL-CFC) media. The number of BL-CFC was counted 4 or 5 days later. The hematopoietic capacity of BL-CFC was detected in methylcellulose culture system and the endothelial potential was assessed by tube-like structure formation on Matrigel. The results showed that the number of BL-CFC in AGM region of Tie2CrePten(loxp/loxp) mouse embryo decreased as compared with Tie2CrePten(loxp/wt) embryo. Whereas the hematopoietic capacity of mutant BL-CFC was enhanced, the endothelial potential, as evaluated by tube-like structure formation in vitro, was significantly reduced. It is concluded that the endothelial PTEN is capable of exerting regulatory functions on both the numbers and the dual potential of hemangioblast in mouse AGM region.

This study was aimed to investigate the effect of RPMI 1640 and IMDM on the development of human peripheral blood monocyte-derived dendritic cells. Under the same cytokines and culture conditions, the different medium types were tested, and the morphology of mature and immature dendritic cells was observed by microscopy, the cell phenotype and endocytosis ability were detected by flow cytometry. Furthermore, the immunoregulatory function of various DC was analyzed by mixed lymphocyte reaction (MLR), the expression of cytokine in culture supernatant of MLR system was also analyzed by Bio-plex technology. The results showed that there were no difference in morphology, CD14, CD83 expression and endocytosis ability between IMDM-cultured DC and RPMI-1640 medium-cultured DC, but there was a lower expression of CD1a in IMDM-cultured DC. Moreover, DC cultured with IMDM displayed a significant reduction in stimulating T cell proliferation, and highly expressed IL-6, IL-8 and IL-10, but low expressed IL-12. It is concluded that the different cultural mediums can induce DC with different functions and DC cultured with IMDM may correlated with induction of immune tolerance. The results of this study will provide a new idea for DC clinical application.

This study was aimed to investigate the effect of human umbilical vein endothelial cells (HUVEC) on dendritic cell (DC) development. First, HUVEC were isolated from human umbilical cord by collagenase digestion, and then the morphology, immunophenotypes and functions were identified. Furthermore, the HUVEC were cocultured with CD14(+) monocytes under the cytokine condition for detecting the influence of HUVEC on differentiation of CD14(+) cells to DC. The phenotype of dendritic cells derived from CD14(+) cells was analyzed by flow cytometry, the immunoregulatory function of DC was tested by mixed lymphocyte reaction (MLR). The change of IL-6 and VEGF as well as EPK and p38 signal pathway were analyzed by neutral antibody experiment and Western blot. The results showed that HUVEC isolated from human umbilical cord were characterized by spindle-shaped morphology, homogenous immunophenotypes (vWF(+)CD31(+)CD73(+)CD45(-)HLA-DR(-)CD86(-)CD34(low)), Dil-Ac-LDL incorporation ability and forming capillary-like structures. Following stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4), HUVEC cocultures could inhibit the initial differentiation of CD14(+) monocyte to DC. Interestingly, IL-6 and VEGF enhanced the suppression effect of HUVEC on generation of DC via activation of the ERK or p38 mitogen activated protein kinase pathway. It is concluded that HUVEC are involved in DC development and can suppress the differentiation of monocyte to DC.

This study was purposed to investigate the effect of mitogen-activated protein kinase (MAPK) pathway on the osteoblast differentiation of mouse mesenchymal stem cells (MSCs), MSCs were isolated from mouse compact bone and serially passaged. After being cultured in osteogenic induction medium, the phosphorylation levels of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 were detected by Western blot. The effects of corresponding pathway inhibitors including PD98059, JNK II and SB203580 on alkaline phosphatase (ALP) and calcium accumulation in the osteoblastic differentiation of MSCs were determined by ALP staining and von kossa staining respectively. The results showed that MAPK pathway including ERK, JNK and p38 was activated in differentiation of MSCs into osteoblasts. ALP activity of MSCs increased in the early phase by addition of PD98059 treatment, whereas ALP activity and calcium accumulation were not observed via JNK II treatment. However, SB203580 strongly inhibited the ALP expression and the calcium accumulation. It is concluded that p38 plays a positive role in the osteogenic differentiation of MSCs, and ERK is probably a negative factor at the early phase of differentiation, but the effect of JNK is not essential.

Leptin, the product of obese gene, is a secreting protein that exerts multiple biological functions by binding to its receptor. Leptin regulates nutrient intake and metabolism, and is secreted from adipocytes, which occupy most of the bone marrow cavity and constitute the microenvironment. Leptin not only plays an important role in the control of the proliferation and differentiation of normal primitive hematopoietic cells, but it also stimulates the growth and viability of leukemic cells. Leukemic cells of some patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia, and chronic myeloid leukemia also express the leptin receptor. Furthermore, leptin also stimulates leukemic cell growth in vivo by promoting angiogenesis. These findings suggest the possibility that leptin and its receptor play roles in the pathophysiology of leukemia, and blockage of leptin binding to its receptor might have potential therapeutic benefits in the treatment of certain leukemias. This review discusses the biological characteristics of leptin and its receptor, the relation of leptin and its receptor with normal hematopoiesis, the relation of leptin and its receptor with AML and so on.

Mesenchymal stem cells (MSCs) are multipotent cells derived from many adult tissues, which can differentiate into cells of the mesodermal lineage, such as adipocyte, osteocyte and chondrocyte, as well as cells of other embryonic lineages. They are a promising tool for tissue engineering. In addition, MSC interacts with immune system, suppressing T cell, B cell and NK cell function and dendritic cell activities. MSC migrates to injured tissue to promote the survival of damaged cells and induces peripheral immune tolerance. The role of MSC in reducing the incidence and severity of graft versus host disease (GVHD) clinically has recently been reported. The immunoregulatory function of MSCs also shows a growing promise in the therapeutic application in autoimmune diseases. This review discusses the mechanism of MSC immunomodulatory ability and its therapeutic potential in autoimmune diseases.

This study was aimed to investigate the effect of activated T cell on the ability of MSC to differentiate into osteoblasts. The activated T cells with MSCs were co-culture for 14 days, then the osteoblast formation was tested by alkaline phosphatase staining. Furthermore, the supernatant of activated T cell was added in culture system of MSCs, the expression of molecules related with immune regulation of activated T cells was detected by RT-PCR, so as to determine what kinds of cytokine displayed the important function in MSC differentiation. The result showed that activated T cell could promote differentiation of MSC into osteoblasts, and IL-1beta played an important role in the effect of activated T cells on MSCs, while TNF-alpha, TGF-beta1 were not. It is concluded that the activated T cells promote the differentiation of MSCs to osteoblasts. The interactive influence between MSCs and immune cells can be mediated through cytokines.

Mesenchymal stem cells (MSCs) are multipotent stem cells which can support hematopoiesis, have immunomodulatory property, may differentiate into osteocytes, chondrocytes and adipocytes, and specifically migrate to damage sites and tumor site, but the mechanism involved in the regulation of migration of MSCs still remains unelucidated. Understanding the fundamental mechanisms underlying MSCs migration holds the promise of developing novel clinical strategies which can deliver antitumor proteins to suppress tumor growth. In this review, the MSC migration in vitro mediated by growth factors, chemokines, adhesion molecules and toll-like receptors are summarized.

This study was purposed to investigate the influence of inflammatory microenvironment on mesenchymal stem cells (MSCs) and regulatory effect of MSCs on osteoblast formation. The MSCs were isolated from synovial fluid of patients with rheumatoid arthritis (RASF-MSCs) and were cultured, the immunotypes of RASF-MSCs were detected by flow cytometry, the ability to differentiate RASF-MSCs into osteoblasts and adipocytes was determined by means of osteogenic and adipogenic induction, the regulatory effect of RASF-MSCs on osteoblast formation was assayed by co-culturing RASF-MSCs whth CD14(+) monocytes and in situ tartrate-resistant acid phosphatase staining. The results showed that RASF-MSCs highly expressed CD105, CD73, CD29, CD44, CD166 and HLA-ABC. Meanwhile, they lowly expressed CD34, CD45, CD31, HLA-DR, CD80 and CD86. However, RASF-MSCs decreased multi-differentiation capability as compared with BM-MSCs. More interestingly, RASF-MSC significantly promoted osteoclasts formation (p < 0.05) when co-cultured with monocytes. It is concluded that MSCs from rheumatoid arthritis synovial fluid exert typical MSC phenotypes but displayed decline of multi-differentiation capability. RASF-MSCs especially show promoting effect on osteoclastogenesis. The findings of this study may contribute to the understanding biological behavior of MSCs in pathological microenvironment.

This study was purposed to clarify whether biology function of mesenchymal stem cells (MSCs) is changed by suppressing the development of dendritic cells (DC) derived from hematopoietic stem cells (HSCs). MSCs were cocultured with dendritic cells derived from CD34 positive hematopoietic stem cells (HSCs), and then the expression of cytokines and phenotypes of DCs/MSCs were detected by RT-PCR and flow cytometry respectively. Induced experiments were used to analyze the differentiation ability of MSCs. The results showed that DCs/MSCs were negative for the CD14, CD34, CD45, CD31, CD86, but positive for HLA-ABC, CD29, CD73, though the percentage decreased as MSCs vs DCs/MSCs (93.1% vs 13.44%, 98.3% vs 78.8%, 95.3% vs 75.9%). In addition, the expression of cytokines such as M-CSF, TGF-beta increased in DCs/MSCs. After differentiation induction, DCs/MSCs were deprived of the potential to differentiate into adipocytes, but maintained osteogenesis characteristics. It is concluded that the basic characteristics of MSCs are altered after coculture with DCs, and DCs/MSCs result in lower expression of mesenchymal phenotypes and decrease differentiation ability, but increase the expression of cytokines related to hematopoiesis and immunity.

Aorta-gonad-mesonephros (AGM) is well known as a main structure that de novo generates hematopoietic primary stem cells (HSC) in mid-gestation mammalian embryos. Hemogenic endothelium, and recently, subendothelial mesenchyme as well as hemangioblast are shown as contributing to blood formation in AGM region. AGM-HSC displays dynamic changes in surface markers, including CD41, CD45 and several endothelial-specific molecules. The novel finding of interleukin-3 as a potent regulator of AGM-HSC seems very interesting. Moreover, zebra fish model reveals PGE2 as a novel stimulator of HSC in AGM and kidney marrow, which is also the case in mouse hematopoietic tissues. Identification of mesenchymal stem cells with significant hematopoietic supporting capacity in AGM region suggests an alternative pathway to explore new molecules governing embryonic and adult hematopoiesis. In this paper, the hemogenic model in AGM region, surface markers on HSCs in AGM region and regulation of HSCs in AGM region were reviewed.

To investigate the effects of microenvironment of aorta-gonad-mesonephros (AGM) on embryonic hematopoiesis, mesenchymal stem cell like stromal cells (MSC like stromal cells) derived from dorsal aorta (DA) in AGM region were separated and identified by their growth characteristics, related molecules expression and mesenchymal lineage potentials. Stromal cells from DA region in mouse embryos (E11.5) were isolated and cultured in vitro. After transfected by pSV3neo-SV40, the clones with G418 resistance were selected, and their growth characteristics were studied. The related molecules were analyzed by flow cytometry, and each clone was induced to differentiate into adipocytes, osteocytes, and chondrocytes. The results showed that most clones (20 clones) selected in the mouse DA region held the morphology of fibroblastoid cells. mDAF3 and mDAF18 could be grown in culture for more than 50 passages with G418 resistance, both have the potential to differentiate into adipocytes, osteocytes, and chondrocytes. At the logarithmic growth period, the cell population doubling time is about 24 hours. Surface markers, such as CD29, CD44, CD105 and Sca-1 were positively detected, while low levels of CD34, CD45, and CD31 were detected. It is concluded that immortalized mDAF3 and mDAF18 have the specific phenotype and differential potency of MSC, which suggests that MSC maybe exist in mouse embryonic DA region, where the MSC like stromal cells can be used as a cell model for further research on the modulation activity of DA microenvironment for embryonic hematopoiesis.

Bone marrow hematopoietic microenvironment occupies the medullary cavities of bones throughout the skeleton and provides support for hematopoiesis and immune cells development. Bone-resorbing osteoclasts in bone marrow environment are specialized cells derived from the hematopoietic stem cells and play a pivotal role in process termed as bone remodeling that involves break down and build-up of bone. It is only recently that studies have provided a novel basis for understanding potential role of osteoclasts in homeostasis, stress-induced mobilization of hematopoietic progenitors and osteoimmunology. Further exploration on the interaction of osteoclasts with others in bone marrow hematopoietic microenvironment may contribute to future clinical treatments for hematopoietic and bone-related immunologic disorders including cancer. In this review the origin and identification of osteoclasts and regulation of mobilizing hematopoietic stem cells, as well as osteoclasts and osteoimmunity were mainly concerned.

Mesenchymal stem cells (MSCs) are a kind of adult stem cells which have the capability to differentiate into multiple cell types as well as self-renew continuously. Recent studies demonstrate that MSCs are low immunogenic and able to exert immunomodulatory function by various approaches, such as suppression of the lymphocyte proliferation, reduction of the dentritic cell generation, maturation and function, down-regulation of the CTL formation and enhancement of regulatory T-cell proportion. In vivo experiments show that MSC infusion can prolong the survival time of allo-skin graft in baboon and ameliorate experimental autoimmune encephalomyelitis in mice. Successful reports have been documented about clinical application of MSC in the management of graft-versus-host disease. In this review, the immunological characteristics and the immunomodulation functions in vitro and in vivo of MSC were summarized.

This study was aimed to investigate whether mesenchymal stem cells (MSCs) existed in human umbilical cord vein and to establish the methods of isolation and expansion in vitro. The MSCs derived for perfusion of umbilical cord vein (UVMSCs) were collected after parturition of Healthy pregnant women. The morphology of MSCs and their differentiation potential into osteoblast, adipocyte and chondrocyte were observed the phenotype and cell cycle of MSCs were determined by using flow cytometry. The result showed that the mesenchymal stem cells separated from umbilical cord vein gave rise to a population of adherent cells with a typical fibroblast-like morphology. Similarly to bone marrow-derived MSCs, they highly expressed CD29, HLA-ABC, CD166, CD105, CD73 and CD44, and were negative for any hematopoietic and endothelial markers (CD45, CD34, CD14 and CD144). Functionally, they could differentiate into the osteoblast, adipocyte and chondrocyte. It is concluded that MSCs exist in the human umbilical cord vein perfusion. Their read amplification in vitro contribute to clinical applications for cell therapy and tissue engineering.

To investigate the possible involvement of Ras signaling in the hematopoietic differentiation of embryonic stem cells (ES cells), ES cells were transfected with RasN17, the dominant-negative mutant of Ras. Western blot was used to test the effect of RasN17 expression on Erk1/2 and Akt phosphorylation, semi-quantitative RT-PCR was used to detect expression of gene related to hematopoiesis in differentiation of ES cells. The results showed that the expression of RasN17 in the ES cells remarkably downregulated the phosphorylation of Erk1/2 and Akt simultaneously. Moreover, the expression of several markers related with hematopoiesis including Runx1, SCL and beta-major globin, were significantly suppressed in the EB expressing RasN17, whereas the transcription of Flk1, a gene required earlier than SCL in development of hematopoietic and endothelial lineages, was not influenced. It is concluded that the activation of Ras is pivotal for in vitro hematopoietic differentiation of ES cells.

To study the regulation of TGF-beta(1) on the development of hemangioblast, embryonic stem cell-derived blast forming cells (BL-CFC) were used as the model of hemangioblast in vitro. TGF-beta(1) or anti-TGF-beta(1) neutralization antibody was added in the medium of embryoid body (EB) generation for observating influence of TGF-B(1) addition in different culture stages on number of BL-CFC and differentiation of BL-CFC to endothelial and hematopoietic cells. The results showed that antagonizing TGF-beta(1) in the course of EB growth could significantly reduce the number of BL-CFC (P<0.01), and the frequency of Flk-1(+) cells was also decreased consistently. Furthermore, the BL-CFC derived from EB pretreated with TGF-beta(1) demonstrated remarkably elevated hematopoietic and endothelial potential, whereas such bi-potential was impaired in the group with neutralizing antibody. It is concluded that TGF-beta(1), a conventional negative regulator in hematopoiesis and angiogenesis exert positive effects on the development and differentiation capacities of BL-CFC in vitro.

This study was purposed to investigate the immunoregulatory effect of endothelial cells derived from mesenchymal stem cells (MSC). The human MSC was induced to differentiate into endothelial cells for one week. The phenotypes were evaluated by flow cytometry, the cell morphologic feature was observed by invert phase-contrast microscope and analysis of capillary formation was performed by using the in vitro angiogenesis kit. The immunoregulatory effect was detected by lymphocyte transformation test. The result indicated that during the differentiation cells grew fast and there was no significant change in the phenotypes, i.e. CD73, CD105, HLA-ABC were positive and CD34, CD80, CD86, HLA-DR, CD31 were negative. Immunofluorescence analysis showed typical expression of the von Willebrand factor. Differentiated MSCs formed capillary-like structure. Endothelial cells derived from MSC also revealed immunosuppressive effect on T cell proliferation in a dose-dependent manner. It is concluded that endothelial cells derived from MSC also harbor immunoregulatory effect on T lymphocytes.

Developmental programs of blood and endothelium are closely correlated and remarkably conserved among species. To categorize the ontogeny relationship between hematopoietic and endothelial lineages, two putative models are presented here. In the yolk sac, hematopoietic and endothelial cells are more likely derived from a common precursor--the hemangioblast. By comparison, the hemogenic endothelium is proposed to characterize the generation of hematopoietic stem cells from mature endothelium in the P-Sp/AGM region. Furthermore, couples of molecules, including Scl, Flk-1 and Runx-1, are involved in formation and subsequent differentiation of the hemangioblast or hemogenic endothelium during embryonic hematopoiesis. In this article, the development of primitive and definitive hematopoiesis, two models associated with development of hematopoietic and vascular endothelial cells as well as molecules associated with development of hematopoietic and endotheliate cells were summaried.

This study was aimed to investigate if human heart harbored a population of primitive undifferentiated cells with the characteristics of MPC. Cells were isolated from human fetal heart and were cultured under conditions appropriate for bone marrow-derived MPCs. Their morphology, phenotypes and functions were tested by methods developed for MPC from other sources. The results showed that morphologically, cells were spindle shaped and resembled fibroblasts. In their undifferentiated state, cells were CD73, CD105, CD29, CD44, HLA-ABC, CD166 positive and CD45, CD34, CD86, HLA-DR negative. When cultured in adipogenic, osteogenic or chondrogenic media, cells differentiated into adipocytes, osteocytes and chondrocytes respectively. They could be extensively expanded in vitro and exhibited very low immunogenicity as evaluated by T cell proliferation assays. It is concluded that cells isolated from fetal heart possess similarity to their adult and fetal bone marrow counterparts in morphologic, immunophenotypic, and functional characteristics.

Mouse embryonic stem cells (ES cells) are pluripotent in that they can give rise to almost all the cell types in vitro and in vivo. Also, they can sustain self-renewal in vitro owing to symmetrical mitosis, i.e., only the cell number increases while the daughter cells remain pluripotent. Self-renewal and pluripotency of ES cells are under stringent regulation of several signaling pathways. Activation of either JAK-STAT3 or PI3K, the downstream cascade of gp130, can maintain the self-renewal of ES cells, while phosphorylation of another gp130-related branch, SHP2-Ras-ERK, drives the differentiation. BMP2/4-mediated signaling is capable of suppressing the differentiation of ES cells in collaboration with activated JAK-STAT3 under serum free culture conditions. Other signaling such as Wnt also contributes to the self-renewal of ES cells. Generally, the network, which is composed of various signaling pathways, modulates the self-renewal and differentiation of mouse ES cells precisely. This review focuses on the role of gp130 in proliferation of mouse ES cells including inhibitory effect of JAK-STAT3 pathway activation on differentiation of mouse ES cells, maintenance effect of PI3K pathway activation on self-renewal of ES cells, promotive effect of SHP-2-Ras-ERK pathway activation on differentiation of ES cells, and influence of other signaling pathways on self-renewal of mouse ES cells, including maintenance effect of BMP combination with LIF under serum free culture conditions on self-renewal of ES cells and promotive effect of Wnt pathway activation on self-renewal of ES cells.

Stem cell growth factor (SCGF) is an early-acting hematopoitic cytokine that has two isoforms including hSCGF with full length molecules and hSCGFbeta, 78 amino acids of which lost in the conserved calcium-dependent carbohydrate-recognition domain (CRD). It has been demonstrated that hSCGFbeta is strictly species-specific in regulating he-matopoiesis. This study was aimed to explore whether human SCGF can exert synergistic stimulatory effect on heterogenous murine CFU-GM progenitor. Firstly, hSCGF cDNA was amplified from human fetal liver cDNA library by using two-step PCR. The hSCGF mature peptide coding sequence was subsequently placed at downstream of glutathione S-transferase (GST) sequence in GST gene fusion expression vector. The results indicated that there existed an additional 60 kD protein compared with mock BL21 when the cells hosting recombinant plasmid were induced with IPTG at 37 degrees C. SDS-PAGE analysis demonstrated that the GST-hSCGF fusion protein mainly existed in insoluble form. When induced at low temperature (28 degrees C), the recombinant protein was mostly soluble. The GST-fusion recombinant protein was subsequently purified by using affinity chromatography. The clonogenic assay revealed that, unlike hSCGFbeta, hSCGF had the granulocyte/macrophage promoting activity (GPA) for murine bone marrow GM progenitor. It is concluded that, in contrast to human SCGFbeta, the intact molecular hSCGF may have no species specificity, implying that CRD domain in human SCGFbeta does not directly bind to corresponding SCGF receptor, but may have certain biological function.

In order to explore what role relative cytokines play in acute GVHD (aGVHD) of mice transplanted with H-2 haploidentical nonmyeloablative bone marrow, a murine model was established by using C57BL/6J mouse as donor and (C57BL/6J x BALB/C) F1 mouse as the recipient. The recipient mice were given CsA and mycophenolate mofetile (MMF) for prophylaxis of aGVHD. The expression levels of IL-2, INFgamma, IL-4 and IL-10 in the spleen were detected by semi-quantitative RT-PCR at different time points after transplantation. The results showed that the expression levels of these cytokines increased slightly in the group only received nonmyeloablative conditioning. The expression levels of IL-2 and INF-gamma elevated significantly after transplantation in group 2 (without aGVHD prophylaxis), peaked at day 21 and day 14 respectively, then dropped rapidly, returned to the basal levels at day 35. The study on kinetic pattern of expression of IL-2 and INF-gamma in group 3 (with aGVHD prophylaxis) gave a similar result to group 2, but the peak value of cytokine expression in group 3 was much lower than that in group 2. The expression of IL-4 in Group 2 and group 3 all peaked at day 14, but the peak value in group 3 was higher than that in group 2, and decreased slowly in group 3. The expression of IL-10 increased gradually after transplantation, peaked at day 14, decreased after day 21, elevated again until day 35. It is concluded that the expression levels of these cytokines in the spleen all increase after H-2 haploidentical nonmyeloablative bone marrow transplantation. CsA and MMF can reduce the incidence and severity of aGVHD by down-regulating the expression levels of IL-2 and INF-gamma.

In an attempt to study the immunoregulatory effect of osteoblasts derived from mesenchymal stem cells (MSC), MSC was induced to differentiate into osteoblasts for one week. The growth pattern and the phenotype were evaluated by MTT and flow cytometry respectively. The immunoregulatory effect was tested by the inhibitory effect on T cell proliferation. The result showed that during the differentiation cells grew fast and there was no significant change in the phenotypes but keeping CD73, CD105, CD44, CD29 positive and CD34, CD45, HLA-DR, CD86 negative. Osteocyte derived from MSC also showed immunosuppressive effect on T cell proliferation in adose-dependent manner. It is concluded that osteoblasts derived from MSC also harbored immunoregulatory effect.

To explore the feasibility of nonmyeloablative conditioning regimens, hematopoietic reconstitution, chimera level and the occurrence of GVHD after nonmyeloablative allogeneic stem cell transplantation in H-2 haploidentical mice, CB6F1 mice were used as the recipient and were divided into 3 groups, mice were pretreated five days before transplantation. Group A was pretreated with myeloablative conditioning regimens (TBI with 10.5 Gy), group B was pretreated by TBI (2 Gy) + Ara-C + Cy and group C-TBI (2 Gy) + Ara-C + CY + Flu, respectively. For all recipient mice, the prevention of GVHD was not given, and 2 x 10(7) bone marrow cells mixed 1 x 10(7) spleen cells from C57BL/6 mice were injected through tail vein on day 0, and then hematopoietic recovery, engraftment and GVHD of recipients were observed. The results of chimera detection after transplantation showed that the engraftment of group A remained full donor chimerism, and engraftments of group B and group C were associated with mixed chimerism or full donor chimerism, but the chimerism of group B remained below 80% and tended to decrease after 50 days whereas chimerism of group C was above 80% (chimerism close to or being full donor type) and preserved even after 50 days. GVHD occurred in all the recipient mice due to that prevention was not given, wherein the occurrence and death rate of GVHD in group A was obviously higher than that of group B and group C (P <0.01), but there was no statistical difference between group B and group C. In conclusion, the nonmyeloablative conditioning regimens mainly based on fludarabine can form stable and lasting engraftment in the body of recipients. The mixed chimerism established in recipients induce tolerance of transplantation and decrease or avoid the occurrence of GVHD.

The objective of this study was to elucidate the effect of human placenta adherent cells (hPDAC) on expansion of human umbilical cord blood CD34(+) cells in vitro. hPDAC was isolated and characterized in human placenta tissue by using enzyme-digesting method and flow cytometry. A co-culture system was established with hPDAC and cord blood CD34(+) cells. The CD34(+) cells were cultured in different culture systems with different combinations of hPDAC and SCF, IL-3, IL-6 and FL. The number of total nucleated cells, CFC and CD34(+) cells were repeatedly counted in culture for 4 weeks. The results showed that the hPDAC displayed fibroblast-like morphology, and were positive for CD29, CD44, CD166, HLA-ABC and UEA-1, and negative for CD34, CD45 and HLA-DR. Functionally, ex vivo expansion of CD34(+) cells on feeder layer of placental adherent cells was significantly higher than that no feeder layer group. SCF + IL-3 + IL-6 + FL + hPDAC manifested the most potent combination, with the number of total nucleated cells increasing by (126.0 +/- 6.7)-fold, progenitor cells (CFC) (49.8 +/- 1.7)-fold and CD34(+) cells (8.3 +/- 1.65)-fold. It is concluded that placental adherent cells could support hematopoiesis in vitro and work as a suitable feeder layer for cord blood stem/progenitor cell expansion.

The purpose of this study was to evaluate whether the DNA vaccine containing idiotypic gene fragment of human B-cell lymphoma cell line Namalwa could elicit the specific anti-idiotypic immune response in vivo. The candidate gene fragment of the lymphoma cell, variable region of heavy chain (VH) of the membranous immunoglobulin, was amplified using Ig superfamily primers by means of RT-PCR. Also, the intact cDNA of murine monocyte chemoattractant protein (MCP-3) was cloned and used as the adjuvant molecular. The two gene fragments of VH and MCP-3 were fused together by 8aa linker peptide with recombinant PCR. Subsequently, the fusion gene fragment was cloned into eukaryonic expression vector pcDNA3.1 to construct DNA vaccine plasmid. Prior to the immunization, the transient transfection coupled with RT-PCR was performed to prove that the recombinant plasmid could express in eukaryonic cells in right way. Then two groups of mice were immunized by intramuscular injection with DNA vaccine and mock plasmid pcDNA3.1 respectively. Three times of injection were performed with 100 micro g plasmid respectively at the beginning of the experiment and 2, 4 weeks after the first injection for all the groups. FACS analysis was chosen to detect the antibodies recognizing lymphoma cells at different time following vaccination. The results demonstrated that specific anti-idiotypic antibody could be detected in the group of DNA vaccine immunized mice as early as eight weeks after the first immunization. Further test demonstrated that the anti-idiotypic antibody could maintain for at least twenty weeks with high titer. Anti-idiotypic antibodies were elicited in three of five mice of the DNA vaccine immunized group. The Abs of DNA vaccine immunized mice could only recognize Namalwa cell line instead of another unrelated human cell line A549. There is no cellular response detected in the DNA vaccine immunized mice. It is concluded that the DNA vaccine containing fused MCP3-VH sequence could elicit specific anti-idiotypic antibody against B-cell lymphoma in vivo and could be used in further study of DNA vaccine against B-cell lymphoma. The results would provide the basis for further studies and optimization of this therapeutic strategy on patients with B-lymphoproliferative disease.

The study was aimed to evaluate if the modified in situ cryopreservation could affect the biological function of mesenchymal stem cells (MSC) in vitro. Mesenchymal stem cells from human bone marrow were isolated by standard method and characterized with their morphology, cell-surface antigen profile and differentiation repertoire in vitro. The culture-expanded MSC were cryopreserved in situ with culture medium (DMEM-LG) containing 10% D MSO and 30% selected FCS in -70 degrees C. Following recovery of cryopreservation, differentiation to adipocytes, chondrocytes, and osteoblast in vitro and cell cycle analysis were performed to investigate whether the cryopreservation would change the differentiation potential of MSC. The results showed that after recovery of cryopreservation, there was no changes detected as compared with the culture-expanded MSC in both differentiation potency and growth pattern at 12 weeks. In conclusions: this optimized short term in situ cryopreservation at -70 degrees C could retain biological characteristics of human MSC for at least 3 months, and this method may be useful for cryopreservation of hum an bone marrow MSCs.

To address the question whether there exists mesenchymal stem cells in adult mouse skeletal muscle, mononuclear cells from muscle were obtained by digestion and density gradient centrifugation and plated in alpha-MEM/F12 medium containing 10% fetal bovine serum. Cell biological properties including morphology, cytochemistry, growth pattern and phenotypes as well were evaluated. Likewise, the osteogenesis of cultured cells was also observed. The results showed that adherent cells homogenous in shape proliferated quickly in the culture system. The phenotypes of the cells were unique, which were positive for CD29 and Sca-1, and negative for CD34 and CD45. Cytochemistry evaluation showed that they were homogeneously positive for acid alpha-naphthl acetate esterase (ANAE) and acid phosphatase (ACP), and negative for alkaline phosphatase (ALP) and that, around 5% of them were positive for glycogen (periodic acid-Schiff reaction, PAS). Cells became ALP-positive after the induction by ascorbic acid, beta-phosphoglycerol and dexamethasone. It is concluded that mesenchymal stem cells exist in murine skeletal muscle and compose the complex heterogenous population of stem cells in muscle.

The blast colony-forming cells (BL-CFC), which are detected within embryoid bodies derived from embryonic stem cells (ES cells) differentiated for 2.5-3.5 days, have dual-potential of differentiation to hematopoietic and endothelial cells. In this investigation the culture method of BL-CFC was established and colony forming assay, immunofluorescent technique as well as nested RT-PCR was employed to identify the differentiation capacity of adherent and nonadherent cells derived from individual blast colony. The results showed that the adherent cells could intake DiI-Ac-LDL and expressed the endothelium-specific surface markers including CD31, UEA-I and VE-cadherin. In addition, nonadherent cells were capable of developing primitive or/and definitive hematopoiesis potential. High proliferative potential colony-forming cells (HPP-CFC) bearing self-renewal capacity was found in 20% of BL-CFC. It is concluded that BL-CFC derived from embryonic stem cells can generate high proliferative potential hematopoietic progenitor cells. However, the whether BL-CFC can reconstitute the adult bone marrow hematopoiesis in long-term remains to be further determined.

In order to research the prophylactic effect of cyclosporine A (CSA) and mycophenolate mofetile (MMF) on GVHD in mice with H-2 haploidentical nonmyeloablative bone marrow transplantation, a murine model was established by using of C57BL/6J mouse as donor and (C57BL/6J x BALB/C) F(1) mouse as the recipient. The recipient mice were given CSA + MTX or CSA + MMF for prophylaxis of acute GVHD (aGVHD). The survival rate, hematopoietic recovery, and morbidity and mortality of aGVHD were observed for 50 days after transplantation. The results showed that typical aGVHD developed in the transplanted mice without prophylactic treatment during 22 to 25 days after transplantation. The morbidity of aGVHD was 75% (15/20), 40% (8/20) and 30% (6/20) and mortality was 100% (15/15), 62.5% (5/8) and 50% (3/6) respectively in unprophylactic group (control), CSA + MTX and CSA + MMF groups. In conclusion, CSA and MTX reduce the morbidity and mortality of aGVHD in mice with haploidentical nonmyeloablative bone marrow transplantation, and the effect of CSA + MMF is better than that of CSA + MTX.

Mesenchymal stem cells (MSCs), precursors of diverse stromal cells, can support hematopoiesis in vitro and can promote the implantation of hematopoietic stem cells in vivo when co-transplanted with CD34(+) cells. The aim of this study was to investigate the potential effect of MSCs on the hematopoietic development of embryonic stem cells (ES cells) and the feasibility of a novel system in which ES cells will be co-cultured with MSCs. The murine bone marrow MSCs were isolated and cultured and then their phenotype and differentiation function were identified with FCM and histochemical technique. The CCE cells, murine ES cell line, were co-cultured with the isolated MSCs and the hematopoietic differentiation of CCE cells was observed with hematopoietic clonogenic assay and RT-PCR. The results showed that the morphology of MSCs became gradually homogeneous with the passage culture of cells. After passage 4, the marker of Sca-1, CD29, CD44 and CD105 were highly expressed, however, CD34 and CD45, the specific marker of hematopoietic and endothelial cells, could hardly be identified. The isolated MSCs differentiated into adipocytes and osteoblasts in specific induction culture system. After maintaining culture on mouse embryonic fibroblasts, CCE cells were plated in suspended culture system with only differentiation inductive agents and co-culture system in which MSCs were added. Compared with CCE cell suspended culture, the cells differentiated into embryoid body were obviously enhanced and there were no colony-forming cells in the co-culture system of ES cells and MSCs. In addition, transcription factor Oct-4 in co-cultured CCE cells was expressed and hematopoietic markers, Flk-1, GATA-1 and beta-H1, were negative. The ability of embryoid bodies derived from the co-culture system to produce hematopoietic colonies was markedly higher than that from the suspended culture system. It is concluded that MSCs inhibit the initial differentiation of ESC and enhance hematopoietic differentiation ability of the co-cultured ES cells.

The object of this study was to explore the effect of FL gene transfected marrow stromal cells on expansion of human cord blood CD34(+) cells ex vivo. A co-culture system was established with FL gene transfected human marrow stromal cells and cord blood CD34(+) cells. The CD34(+) cells were cultured in different culture systems with different combinations of stromal cells and SCF, IL-3 and FL. The numbers of total nucleated cells, CFCs and CD34(+) cells were repeatedly counted in culture systems for 3 weeks. The results showed that the FL transgenic marrow stromal cell feeder layer significantly enhanced the expansion of total nucleated cells, CFC and CD34(+) cells, compared with stromal cell feeder layer and stromal cell-free culture systems. The transgenic stromal cells and cytokines co-culture system manifested the most potent expansion of the cord blood cells with increase of the number of total nucleated cells by (122.5 +/- 4.3)-fold, CFCs by (39.6 +/- 2.7)-fold and CD34(+) cells by (11.8 +/- 0.52)-fold. It is concluded that human umbilical cord blood CD34(+) cells can be extensively expanded ex vivo by using FL gene transfected stromal cells combined with cytokines.

Embryonic hematopoiesis in mammals is characterized by successive temporal and spatial changes. Previous investigations indicate that in vitro differentiation of embryonic stem cells (ES cells) derived from 129 mice can mimic embryonic hematopoiesis to some extent. To investigate the in vitro hematopoietic differentiation capacity of ES cells derived from C57BL/6 mice, the authors initially established the murine ES cell line with standard identification methods employed. Next, two-step culture system was utilized for embryoid bodies formation and the appearance of different hematopoietic precursors was confirmed by CFC assay, cellular chemical staining as well as RT-PCR. The results demonstrated that the ES cell line MES-1 fulfilled the criteria of ES cell line and its progeny after in vitro differentiation included primitive and definitive erythrocyte precursors, mixed colony-forming cells and granulocyte/macrophage colony-forming cells. RT-PCR analysis revealed the molecular consistence of transcription factors and hematopoietic markers with cellular event. In conclusion, MES-1 established from C57BL/6 mice was able to differentiate in vitro to a variety of hematopoietic precursors, thus could partly recapitulate embryonic hematopoiesis.

To study the effect of mesenchymal stem cell (MSC) on immune function, MSCs were isolated and cultured from human bone marrow cells. The purity of MSCs were identified with the spindle-fibroblastic morphology characterization by microphotograph and the phenotypes were tested by flow cytometry. MSCs were plated in 96-well plates (2,000/well and 1,000/well), and cocultured for 3 days with T cells isolated from cord blood. Cord blood T cells non-cocultured with MSC acted as control group. After cord blood T cells stimulated by PHA for 60 hours, [(3)H]-thymidine was added to each well and T cell proliferation was assessed by [(3)H] thymidine incorporation. The results showed that cord blood T cell proliferation was suppressed when 2,000 MSCs were plated each well and cord blood T cell proliferation was activated when 1,000 MSCs were plated. Our results suggested that the immunomodulatory function of MSC seemed dependent on cell dose. High concentration of MSC most often resulted in inhibition, while low concentration resulted in stimulation.

To investigate the mechanisms underlying the issue that bone marrow mesenchymal stem cells (MSC) could trigger low responsiveness of allogeneic T lymphocytes against alloantigens, phenotypes of T cells were analysed with flow cytometry before and after coculture of allogeneic T lymphocytes with MSC. Further, expression of cytokines including IL-4, IFN-gamma and TGF-beta1 was evaluated by RT-PCR and ELISA as well. The results showed that CD8(+) subpopulation was increased and CD25(+) subset was decreased in proportion after coculture of allogeneic T lymphocytes with MSC for 2 weeks. RT-PCR assay showed that MSC expressed TGF-beta1 but not IL-4 and IFN-gamma, and the result was further proved by ELISA assay showing that the secretion of TGF-beta1 reached to 1 ng/ml in 72 hours. It was concluded that allogeneic MSC suppressed T cells activation by alteration of T cell subpopulations. The suppressive effect was at least in part due to the secretion of TGF-beta1. The results indicate that MSC pretreatment might be useful in the prevention of GVHD in HLA-mismatched bone marrow transplantation and further donors for hematopoietic stem cells could be selected from greater potentials.

In order to reach the purpose of co-transferring double drug resistance genes into human CD34(+) progenitor cells to broaden the spectrum of drug resistance, the expression efficiency of human multidrug resistance 1 (MDR1) gene mediated by the internal ribosomal entry site (IRES) was investigated. Two retroviral vectors were transferred into packaging cells. One is pSF-DIM containing double drug resistance genes, in which the translation of MDR1 gene was controlled under an IRES from encephalomyocarditis virus. The other is pSF-MDR1 which only contains MDR1 gene controlled under the same promoter of pSF-DIM. The amphotropic retroviral packaging cells PA317/pSF-DIM and PA317/pSF-MDR1 were obtained with titer of 8 x 10(4) and 1.3 x 10(5) cfu/ml respectively. Human cord blood CD34(+) cells were transduced by supernatant infection. Expression of P-gp was detected by flow cytometry. Compared with the untransduced group, the expression of P-gp in pSF-DIM transduced group and pSF-MDR1 transduced group was elevated 10.92% and 28.82% respectively. However, the expression of P-gp in pSF-MDR1 transduced group was higher than that in pSF-DIM transduced group. The result suggests that MDR1 gene can express in the human progenitor cells under control of IRES. It laid the foundation of subsequence research. The reason on the difference in MDR1 gene expression efficiency between pSF-MDR1 transduced group and pSF-DIM transduced group need further research.

CD40/CD40L interactions play a pivotal role in T cell activation, and take part in many physiologic and pathologic procedures and different levels. In this article, stable CHO transformants secreting human CD40-Ig fusion protein were established through transfection and selection with Lipofectamaine and G418, respectively. In order to obtain great valume of recombinant protein, big batch serum-free cultures of engineered CHO cells were performed in roller-bottle using CHO-II-SFM medium. After cultures, the cell-culture supernatants were harvested, concentrated through ultra-filtration, and finally purified by affinity choromatography with Protein G Sepharose Fast Flow. Human peripheral bloods were collected freshly and seperated with Ficoll, CFU-T was cultured in semi-solid culture system with peripheral blood mononuclear cells (PBMNC). Effect of human CD40-Ig fusion protein on the formation of CFU-T was observed in vitro. The results showed that the yield of human CD40-Ig fusion protein was 30 mg in total 3 liter CHO-II-SFM culture supernatant, and it supposed that the expression level of CD40-Ig in CHO cells was more than 10 micro g/ml. The purity of purified fusion protein is above 95%. Furthermore, compared with human IgG, human CD40-Ig fusion protein significantly inhibited the formation of CFU-T at dose 0.25, 1.0, 4.0, and 10 micro g/ml, it lays a good foundation to evaluate its potential functions in vivo.

In the present study, an attempt was made to prove the question whether endothelial cell precursors exist in blood circulation during postnatal period. CD34(+) cells were harvested from G-CSF mobilized adult blood and umbilical cord blood and incubated onto fibronectin/gelatin-coated Petric dishes in the presence of recombinant human vascular endothelial cell growth factor(rhVEGF) and basic fibroblast growth factor(rhbFGF). Endothelial cell lineage was identified by von Willebrand factor(vWF) expression and Ulex europous agglutinin I(UEA-I) binding capacity. The results showed that a firmly adherent cell monolayer formed when CD34(+) cells, but not CD34(-) cells, were cultured for 5 - 6 weeks as described before. Immunocytochemistry and flow cytometry analysis showed that almost all of the adherent cells were vWF-positive and around 90% were able to bind UEA-I specifically. These findings demonstrate that angioblasts exist in the circulation during postnatal life and therefore, vasculogenesis might occur in adults.