AIMTo observe the basical properties of adherent stromal cells in culture derived from cryopreserved bone marrow cells (BMCs), and to provide laboratory evidences for clinical application of cryopreserved stromal cells.

METHODSFresh BMCs and adherent stromal cells cultured for 14 days in Dexter long-term culture system (fresh stromal cells) plus 5% DMSO-6% HES cryopreservatives were frozen in -80 degrees C refrigerator, and cryopreserved in - 196 degrees C liquid nitrogen for 2 weeks (the former is called cryopreserved BMCs, the latter called cryopreserved stromal cells). These cells were cultured in Dexter long-term culture system after they were thawed. We have examined the growth features, constituents and stimulating functions of the culture of these cells.

RESULTSGrowth features: Cryopreserved BMCs produced adherent stromal cells, cell clusters and cell layer 1-2 days later than fresh BMCs. Cryopreserved stromal cells formed cell layer in 2nd day of culture, and were 12-18 h later than fresh stromal cells. Cryopreserved BMCs and stromal cells proliferated significantly less than fresh BMCs and stromal cells. Constituents: The ratio of fibrocytes and endothelial cells were lower, and the ratio of macrophages and fat cells were higher in culture of cryopreserved BMCs than that in fresh BMCs. The measurements in culture of cryopreserved stromal cells were much more significant compared with that in fresh stromal cells. Numbers of cells containing apoptic bodies in culture of cryopreserved BMCs and stromal cells were more than that in fresh BMCs and stromal cells. TBRR of cryopreserved BMCs and stromal cells were 92.5% and 89.5% respectively. The expression rates of CD14 and HLA-DR in culture of cryopreserved BMCs and stromal cells were higher than that in fresh BMCs and stromal cells, and just in contrast with the expression rates of CD45 and CD33. Stimulating functions: CAFA and LTC-IC on the stromal cell layer derived from cryopreserved BMCs, cryopreserved stromal cells, fresh BMCs and fresh stromal cells were all growing well, and there were no significant differences among these groups.

CONCLUSIONBiological properties of adherent stromal cells derived from BMCs and stromal cells were injured slightly and still maintained completely after cryopreservation with 5% DMSO-6% HES cryopreservatives.

This study was to establish an iron overload bone marrow (BM) model by co-culturing the mononuclear cells from BM with iron, and investigate its hematopoiesis changes. The iron overload model was set up by adding different concentration of ferric citrate (FAC) into the mononuclear cells from BM and culturing for different time, and the model was confirmed by detecting labile iron pool (LIP). Then the apoptosis of hematopoietic cells, ability of hematopoietic colony forming (CFU-E, BFU-E, CFU-GM and CFU-mix) and percentage of the CD34(+) cells of the BM cells all were determined. The changes of these indexes were tested after the iron-overloaded BM was treated with deferasirox (DFO). The results showed that after BM cells were cultured with FAC at different concentrations for different time, the LIP increased in time-and concentration-dependent manners. The intracellular LIP reached maximum level when cultured at 400 µmol/L of FAC for 24 hours. The detection of BM cell hematopoietic function found that the apoptotic rate of the FAC-treated cells (24.8 ± 2.99%) increased significantly, as compared with normal control (8.9 ± 0.96%)(p < 0.01). The ability of hematopoietic colony forming in FAC-treated cells decreased markedly, as compared with normal control (p < 0.05). The percentage of CD34(+) cells of FAC-treated cells (0.39 ± 0.07%) also decreased significantly, as compared with normal control (0.91 ± 0.12%)(p < 0.01). And these changes could be alleviated by adding DFO. It is concluded that the iron-overloaded model has been set by adding iron into the mononuclear cells from BM in vitro, and the hematopoietic function of iron-overloaded BM is deficient. These changes can be alleviated by removing the excess iron from the BM cells through treating with DFO. These findings would be helpful to further study the mechanism of iron-overload on the hematopoiesis of BM and also useful to find the way to treat iron-overload patients with hematopoietic disorders.
Bone Marrow Cells ; cytology ; Cells, Cultured ; Hematopoiesis ; Hematopoietic Stem Cells ; cytology ; Humans ; Iron ; metabolism ; Iron Overload

OBJECTIVETo review the in vitro development of bone marrow mesenchymal stem cells culture (BM-MSC).

DATA SOURCESThe data cited in this review were mainly obtained from articles listed in Medline and PubMed. The search terms were "bone marrow mesenchymal stem cell" and "cell culture".

STUDY SELECTIONArticles regarding the in vitro development of BM-MSCs culture, as well as the challenge of optimizing cell culture environment in two-dimensional (2D) vs. 3D.

RESULTSImproving the culture conditions increases the proliferation and reduces the differentiation. Optimal values for many culture parameters remain to be identified. Expansion of BM-MSCs under defined conditions remains challenging, including the development of optimal culture conditions for BMSC and large-volume production systems.

CONCLUSIONSExpansion of BM-MSCs under defined conditions remains challenges, including the development of optimal culture conditions for BMSC and scale-up to large-volume production systems. Optimal values for many culture parameters remain to be identified.

Therapeutic angiogenesis is a promising new method for the treatment of ischemic heart disease, and in recent years, implantation of autologous bone marrow cells has opened up a new pathway to such object and is now attracting many researchers' attention. This article analyzes the cell biological base of autologous bone marrow cells and the possible mechanism of neovascularization induced by implantation of autologous bone marrow cells; in addition, the effect, safety and feasibility of the implantation of autologous bone marrow cells are reviewed and commented on the basis of recent researches in animal and clinical trials.
Animals ; Bone Marrow Cells ; cytology ; Bone Marrow Transplantation ; Humans ; Myocardial Ischemia ; therapy ; Neovascularization, Physiologic

OBJECTIVETo explore effects of iron overload on hematopoiesis in mice with bone marrow injury and its possible mechanism (s).

METHODSC57BL/6 mice were divided into control, iron, irradiation, irradiation+iron groups. The iron-overloaded model of bone marrow injury was set up after mice were exposed to the dose of 4 Gy total body irradiation and (or) were injected iron dextran intraperitoneally. Iron overload was confirmed by observing iron deposits in mice and bone marrow labile iron pool. Additionally, the number of peripheral blood and bone marrow mononuclear cells and the frequency of erythroid cells and myeloid cells were counted and hematopoietic function was assessed.

RESULTS(1)Iron overload occurred by bone marrow biopsy and flow cytometry analysis. (2)Compared with control group, the number of platelets [(801.9±81.2)×10⁹/L vs (926.0±28.2)×10⁹/L] and BMMNC and the frequency of erythroid cells and myeloid cells decreased. Moreover, hematopoietic colony forming units and single-cell cloning counts decreased significantly in irradiation group (P<0.05). (3)Compared with irradiation group, the number of platelets [(619.0±60.9)×10⁹/L vs (801.9±81.2)×10⁹/L] and the frequency of erythroid cells and myeloid cells decreased; moreover, hematopoietic colony forming units and single-cell cloning counts decreased significantly in irradiation+iron group (P<0.05). (4)Compared with irradiation group, ROS level increased by 1.94 fold in BMMNC, 1.93 fold in erythroid cells and 2.70 fold in myeloid cells, respectively (P<0.05).

CONCLUSIONThe dose of 4 Gy total body irradiation caused bone marrow damage and iron overload based on this injury model, which could damage bone marrow hematopoietic function aggravatingly. And further study found that iron overload was closely related to increased ROS level in BMMNC. The findings would be helpful to further study the injury mechanism of iron overload on the hematopoiesis of bone marrow.

Mesenchymal stem cells (MSCs) could be obtained from many sources, and there are differences between them. This study was purposed to compare and analyze the basic biological characteristics of umbilical cord, adipose tissue-and bone marrow-derived MSC (UC-MSCs, AD-MSCs and BM-MSCs). The MSCs were isolated from umbilical cord, adipose tissue and bone marrow were cultured; the morphology of UC-MSCs, AD-MSCs and BM-MSCs was observed by using microscopy; the immunophenotype, differentiation potential and expression of peroxisome proliferation-activated receptor-γ (PPAR-γ) mRNA were detected by using flow cytometry, differentiation test (von kossais and 0:1 red O staining) and quantitative fluorescent PCR, respectively. The results showed that the UC-MSCs, AD-MSCs and BM-MSCs displayed similar morphology under confocal microscope after being stained with rhodamine phalloidin and DAPL. The immunophenotypes of these three originated cells conform to coincide with identification criterion for MSCs, and showed similar expression level. During adipogenic induction the adipogenic potential of these MSCs was different, AD-MSCs exhibited the highest adipogenic potential, UC-MSCs displayed the lowest, while potential of BM-MSCs get between; however, the osteogenic differentiation potential of UC-MSCs, AD-MSCs and BM-MSCs was similar. The PCR detection showed that the expression level of PPAR-γ mRNA was the highest in AD-MSCs and the lowest in UC-MSCs, while expression level in BM-MSCs get between, these results were identical with the adipogenic potential, suggest that the difference of adipogenic potential in 3 kinds of MSCs was associated with basic expression level of PPAR-γ mRNA. It is concluded that UC-MSCs, AD-MSCs and BM-MSCs exhibit similar morphology, the immunophenotypes of these MSCs coincide with identification criterion for MSCs, the osteogenic potential of these MSCs is similar, while the adipogenic potential and the expression level of PPAR-γ mRNA are different. The difference-associated mechanisms need to further study.
Adipogenesis ; Adipose Tissue ; cytology ; Bone Marrow Cells ; cytology ; Cell Separation ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; Umbilical Cord ; cytology

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.
Arthritis, Rheumatoid ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; Osteoblasts ; cytology ; Synovial Fluid ; cytology

Bone marrow mensenchymal stem cells (BM-MSCs) are capable of supporting the survival, differentiation and migration of hematopoietic stem cell, and have a profound application prospect in transplantation and treatment of graft-versus-host disease (GVHD). This review aims to illustrate the interaction between BM-MSCs and other intra-bone marrow cells, including hematopoietic stem cells, endothelial cells and osteoblasts. The investigation of their regulating mechanism will help better understanding of the BM-MSCs' role in hematopoiesis.
Bone Marrow Cells ; cytology ; Cell Communication ; physiology ; Endothelial Cells ; cytology ; Humans ; Mesenchymal Stromal Cells ; cytology ; Osteoblasts ; cytology

Strong proliferative capacity and the ability to differentiate into the derivative cell types of three embryonic germ layers are the two important characteristics of embryonic stem cells. To study whether the mesenchymal stem cells from human fetal bone marrow (hfBM-MSCs) possess these embryonic stem cell-like biological characteristics, hfBM-MSCs were isolated from bone barrows and further purified according to the different adherence of different kinds of cells to the wall of culture flask. The cell cycle of hfBM-MSCs and MSC-specific surface markers such as CD29, CD44, etc were identified using flow cytometry. The expressions of human telomerase reverse transcriptase (hTERT), the embryonic stem cell-specific antigens, such as Oct4 and SSEA-4 were detected with immunocytochemistry at the protein level and were also tested by RT-PCR at the mRNA level. Then, hfBM-MSCs were induced to differentiate toward neuron cells, adipose cells, and islet B cells under certain conditions. It was found that 92.3% passage-4 hfBM-MSCs and 96.1% passage-5 hfBM-MSCs were at G(0)/G(1) phase respectively. hfBM-MSCs expressed CD44, CD106 and adhesion molecule CD29, but not antigens of hematopoietic cells CD34 and CD45, and almost not antigens related to graft-versus-host disease (GVHD), such as HLA-DR, CD40 and CD80. hfBM-MSCs expressed the embryonic stem cell-specific antigens such as Oct4, SSEA-4, and also hTERT. Exposure of these cells to various inductive agents resulted in morphological changes towards neuron-like cells, adipose-like cells, and islet B-like cells and they were tested to be positive for related characteristic markers. These results suggest that there are plenty of MSCs in human fetal bone marrow, and hfBM-MSCs possess the embryonic stem cell-like biological characteristics, moreover, they have a lower immunogenic nature. Thus, hfBM-MSCs provide an ideal source for tissue engineering and cellular therapeutics.
Bone Marrow Cells ; cytology ; Cell Proliferation ; Embryonic Stem Cells ; cytology ; Fetus ; Germ Layers ; cytology ; Humans ; Mesenchymal Stromal Cells ; cytology

OBJECTIVENoting the morphological and cytobiology characteristics and phenotypes of MMSCs, to establish an isolation and culture method for fetal MMSCs in order to provide a source of marrow mesenchymal stem cells (MMSCs).

METHODSFetal MMSCs were isolated and cultured with in vitro cell culture technique; the characteristics of the proliferating and growing fetal MMSCs were studied with MTT and image analysis; the phenotypes of MMSCs were identified by flow cytometry and immunohistochemistry.

RESULTSBone marrow of 12 fetuses was isolated within 0.5-2 h, and about 300+/-80 adherent cells were obtained at 24 h. Colonies with more than 5 cells were 15+/-6, growth detention period of culture cell was at 1-3 d after planting, log phase growth period was at day 4, and the amount of disintegration phase cells was reduced significantly. Original culture and serial subcultivations showed that cells divided prosperiously; unequal divisions special for stem cells were observed, and the amount of MMSCs harvested from each fetus was as much as 10(11)-10(12) cells after 10 serial subcultivations. The phenotype of MMSCs was CD166 positive and CD34 negative. Serial subcultivated MMSCs expressed a microamount of AFP and did not express albumine or CK18.

CONCLUSIONFetal MMSCs are easily isolated and proliferate prosperouly. Serial subcultivated MMSCs did not differentiate into hepatocyte-like cells under common culture condition and are feasibile as seed cells for tissue engineering reconstruction.