Osteoblasts derived from mesenchymal stem cells harbor immunoregulatory effect / 中国实验血液学杂志

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.

Comparison of mesenchymal stem cells from human placenta and bone marrow / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Nowadays 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.</p><p><b>METHODS</b>Mononucleated 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).</p><p><b>RESULTS</b>Human 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.</p><p><b>CONCLUSION</b>These 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.</p>

Current basic research of hematopoietic stem cells in China and comments on stem cell plasticity / 中国实验血液学杂志

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.

Human placenta derived adherent cells support in vitro expansion of umbilical cord blood CD34+ cells / 中国实验血液学杂志

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.

More on Hematopoietic Stem Cell Research and Application Updated / 中国实验血液学杂志

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.

Maintaining Growth of Long-Term Culture Initiating Cells from Human Cord Blood on Feeder Layers of Bone Marrow Stromal Cells Transfected with FL and/or TPO Genes / 中国实验血液学杂志

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.

The Presence of Endothelial Cell Precursors in Blood Circulation / 中国实验血液学杂志

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.

Challenges to Hematopoietic Stem Cell Research Today / 中国实验血液学杂志

More works documented recently indicated that human CD34(-) cells exist and are likely to be the precursors of the CD34(+) cells. Anyhow, the CD34(+) enriched populations have already been proved to show long-term reconstitution of hematopoiesis in animals and patients worldwidely. It still remains uncertain whether cells lack of CD34 and Lineage markers are the very best stem cells or maybe the residual embryonic stem cells keeping quiescent in the adult tissues are capable of transfer into hematopoietic stem cells when activated. Since just a negative selection technique is used to collect the CD34(-) cells everywhere for the time being, no final conclusion is convincing about the characterization of CD34(-) cells till a highly purified cell population of CD34(-)/Lin(-) is available. Clinical analysis shows that the most critical factor predicting the stem cell engraftment is the number of the cells infused. The number of nucleated cells in umbilical cord blood to be infused and required to obtain a successful engraftment is superior to 3.7 x 10(7)/kg. However, large dose of T-cell-depleted and purified CD34(+) cells as more than 5 x 10(6)/kg or even a 'megadose' of CD34(+) cells of 10(7)/kg is recommended for allotransplant of mobilized peripheral blood to achieve a high rate of successful engraftment. The delayed engraftment and the relapse of malignancy after cord blood transplantation are major problems. However, CBT is still the best choice of stem cell transplant for the baby patients with non-malignancies. At present, the HLA typing for class I antigens is still achieved with serology in most laboratories. As HLA typing is increasingly defined to higher degree of resolution by DNA probes, it is recommended to check with genotyping when there is a 'match' by the serological phenotyping especially for the unrelated donor/recipient couples. Improvements in DNA-based methods for the detection of numerous HLA alleles have provided the opportunity to investigate the relationship between HLA disparity and transplant complications. About 80% (or even more) of patients in China who might benefit from stem cell transplantation still fail to find suitable donors. It is worthy to adopt the unrelated donors matched or mismatched for those high-risk acute leukemia patients who do not have related matched donors but urgently need transplant. The advanced experience of unrelated mismatched transplant will no doubt be certain to carry weight and be disseminated in China. A great leap forward of the clinical practice and biological study on hematopoietic stem/progenitor cells is expected in the new century to accept the challenge from the world outside China.

Construction of Expression Vector for Human CD40-Ig Fusion Protein and Its Expression in COS-7 Cells / 中国实验血液学杂志

CD40/CD40L, besides B7/CD28, is an alternative important costimulation signal transduction pathway. It plays a pivotal role in T cell activation. Moreover, it may play a critical role at many levels of sensitization and effector phases of allograft rejection. In order to get the fusion protein of human CD40 extracelluar region and IgG 1 Fc fragment, and investigate the potential role of blocking CD40/CD40L costimulation pathway in immunotherapy, total RNA was extracted from human lymphoma cell line Daudi, and CD40 gene extracelluar region was amplified by RT-PCR. The PCR products were inserted into pGEM T Easy vector, and the cloning vector pGE40 was obtained. The DNA sequence was analyzed by automatic DNA sequencer. After sequencing, the transient expressing vector was constructed by inserting correct fragment into pIG vector, which contains the genomic human IgG1 Fc (hinge, CH2 and CH3) gene. Hence the recombinant fusion expression vector was constructed successfully, and named after pIG/40 Ig. Then, COS-7 cells were transfected through DEAE-Dextran/chloroquine method. The CD40-Ig fusion protein expressed in COS-7 cell culture supernatant was identified by sandwich ELISA and Western blot. Result showed that the CD40-Ig fusion protein can be detected by sandwich ELISA in the cell culture supernatant. Western blot analysis also showed that it could react with McAbs of mouse anti-human CD40 G28-5 and mouse anti-human Ig gamma chain. There is only one obvious band at the position of relative molecular weight 50 kD, and it is equivalent to the expected value. Above all, the recombinant fusion expression vector pIG/40 Ig was constructed, and CD40-Ig fusion protein gene was expressed in COS-7 cells successfully. It could be laid a foundation to investigate the potential role of CD40/CD40L pathway as the target of GVHD prevention and therapy.