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.
Bone Marrow Cells ; cytology ; immunology ; Cell Differentiation ; immunology ; Cell Lineage ; Cell Proliferation ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; immunology ; Osteoblasts ; cytology ; immunology ; T-Lymphocytes ; cytology ; immunology

BACKGROUNDNowadays bone marrow represents the main source of mesenchymal stem cells (MSCs). We identified a new population of MSCs derived from human placenta and compared its biological characterization with bone marrow derived MSCs.

METHODSMononucleated cells (MNC) were isolated from the human placenta tissue perfusate by density gradient fractionation. Individual colonies were selected and cultured in tissue dishes. At the same time, human bone marrow derived MSCs were identified. Culture-expanded cells were characterized by immune phenotyping and cultured under conditions promoting differentiation to osteoblasts or adipocytes. The hematopoietic cytokines were assayed using reverse transcriptase polymerase chain reaction (RT-PCR).

RESULTSHuman placental MSCs exhibited fibroblastoid morphology. Flow cytometric analyses showed that the placental MSC were CD29, CD44, CD73, CD105, CD166, HLA-ABC positive; but were negative for CD34, CD45, and HLA-DR. Functionally, they could be induced into adipocytes or osteocytes. Moreover, several hematopoietic cytokine mRNA was found in placenta-derived MSCs by RT-PCR analysis, including IL-6, M-CSF, Flt3-ligand and SCF. These results were consistent with the properties of bone marrow derived MSCs.

CONCLUSIONThese observations implicate the postpartum human placenta as an important and novel source of multipotent stem cells that could potentially be used for investigating mesenchymal differentiation and regulation of hematopoiesis.

Bone Marrow Cells ; physiology ; Cell Differentiation ; Cells, Cultured ; Female ; Humans ; Mesenchymal Stromal Cells ; physiology ; Placenta ; cytology ; Pregnancy

Adult ; China ; Humans ; Mesenchymal Stem Cell Transplantation ; Research

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.
Antigens, CD34 ; analysis ; Cell Adhesion ; Cell Separation ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans ; Placenta ; cytology

The basic studies selected were mainly published since 1998 and related to stem cell biology and engineering and particularly the efforts for developing new sources of hematopoietic stem/progenitor cells ex vivo. Hematopoietic cells and lymphocytes can be developed by induced differentiation in a appropriate way of culture, originating in the embryo- or adult-derived stem cells or tissue-committed stem cells which still exist in the tissue of adults. The most primitive multipotential embryonic stem cell from embryo or adult tissue has the plasticity to differentiate into every kind of progenies, the committed tissue-specific stem cell, by different proper ways of induction in vitro. The committed tissue-specific stem cell, however, can only be induced to differentiate along the line of its committed origin of tissue. No studies in China strongly confirmed yet the existence of "transdifferentiation" among the tissue- or organ-specific stem cells.
Adult ; Cell Differentiation ; Cell Lineage ; China ; Embryo, Mammalian ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans ; Mesoderm ; cytology ; Models, Biological ; Pluripotent Stem Cells ; cytology ; Research ; trends ; Stem Cells ; cytology

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.

Long-tem culture initiating cells(LTC-IC), and in vitro assay of hematopoietic stem/progenitor cells, still represent a heterogeneous population in terms of proliferative capacity and sensitivity to different growth factors. Human umbilical cord (CB) is rich of hematopoietic progenitor cells measured by clonogenic assays and stem cells capable of reconstituting the marrow after transplantation. The influence of culture conditions on the in vitro behavior of LTC-IC from CB was evaluated. First, by using IRES sequence, FL and TPO cDNA were recombined with retroviral vector pLXSN by gene recombination technology. The recombinant plasmid pLFSN, pLTSN, pLFTSN were transfected into human stromal cell line HFCL. Then, LTC-IC were evaluated in long term cultures, comparing five types of stromal feeder layers: human bone marrow stromal cell, human stromal cell line HFCL, and stromal cell lines HDF tranfected with FL gene, HLT transfected with TPO gene or HFT co-transfected with FL and TPO genes. The results were demonstrated that after 8 weeks of coculture, three types of stromal cell lines that supported the maintenance of CFU-C for up to 3 weeks in vitro were identified. However, cocultivation of human bone marrow stromal cell and CB CD34(+) cells on HFT, CFU-C production continued up to 6 weeks or longer on these stroma. The absolute LTC-IC frequency in CD34(+) cells on human bone marrow stromal cell (2.65 +/- 0.76/1 000 cells) was no significant difference with on HFT (3.65 +/- 0.58/1 000 cells). Thus, HFT acts by direct contact to maintain the phenotype and function of the most primitive and quiescent human progenitors. Furthermore, HFT cell line was selected as the optimal one for supporting long-term culture feeder. It was concluded that LTC-IC progenitors from cord blood maintain growing upon the FL/TPO gene-modified stromal feeder layers in vitro.

Allogeneic hematopoietic stem cell transplantation (HSCT) is an effective and proven treatment for malignant and nonmalignant diseases. Most of the natural HSC allografts hardly show overall advantages of high engraftment, slight GVHD and rare relapse. The graft engineering including stem cell engineering to make a tailor-made graft ex vivo is promising to conquer all the risks of low engraftment, lethal GVHD and high relapse, which becomes the key program in current HSC research. To combine HSC allotransplant with gene therapy and immune therapy is the novel therapeutic strategy for malignancies, the real meaning of "cytotherapy" or "cell therapy" updated. The rapid expansion of umbilical cord blood banks (CBBs) makes the substantial increase of cord blood transplants (CBT) both possible and likely world-widely. In China, however, owing to lack of hematological pediatricians specified in HSCT and pediatric laminar-flow wards, clinical application rate of cord blood is extremely low despite of the high collection rate in the CBBs under the GMP standards. In evaluating a CBB, the release rate and the clinical efficacy of the released cord blood should be most emphasized as well as the banking quality control. In all CBBs worldwide, it is unworthy of mentioning the banking of umbilical cord blood for autologous transplant. Only one or two commercial companies in the world run it for profitable purpose to charge the donor parents regularly. It is because no autologous CBT can cure the inherited diseases and its efficacy of treating malignancies is doubtable since the cord blood is of weak immune competence against tumor and may be contaminated with autologous malignant or premalignant cells. Moreover, there is no report so far about how long the repopulating activity of cryopreserved hematopoietic stem/progenitor cells of cord blood can keep. No honest guarantee can be made about the effective quality and adequate amount of stem cells to meet the therapeutic requirement when used after a long storage.

Abstract:Objective To investigate the protective effects of blocking CD40/CD40L interactions with human CD40-Ig fusion protein in a murine graft-versus-host disease model.Methods Human CD40 gene extracellular region was inserted into plasmid pIG1, which contains genomic human IgG1 Fc gene. A transient vector containing CD40-Fc fusion gene was transfected into COS-7 cells. The CD40-Ig fusion protein was detected through enzyme-linked immunosorbent assay (ELISA). A constitutive vector was also generated by ligating the CD40-Fc fusion gene into pcDNA3.1 and transfecting it into CHO cells. CD40-Ig was purified by protein A affinity chromatography. SDS-PAGE, Western blot and ligand binding assay were used to identify the qualities of CD40-Ig. Murine acute graft-versus-host disease (GVHD) was induced by intravenous injection of C57BL/6J (H-2b) spleen cells into sub-lethally irradiated BALB/c (H-2d) mice. Protective effects against murine graft-versus-host disease by in vivo administration of CD40-Ig were evaluated.Results Mammalian expression vectors pIG/40Ig and p3.1/40Ig were constructed as described above. Chimeric proteins were expressed in COS-7 and CHO cell culture supernatant and confirmed by ELISA and Western blot. SDS-PAGE showed that fusion proteins had a disulfide-bonded dimeric structure and existed as homodimer. Purified CD40-Ig could bind to CD40L. In vivo administration of CD40-Ig could prevent the development of GVHD and significantly prolong the mean survival time of mice with graft-versus-host disease.Conclusions These results demonstrate that CD40/CD40L interactions play an important role in the pathogenesis of graft-versus-host disease and suggest clinical potential for CD40-Ig in the prevention and treatment of human graft-versus-host disease.