A series of studies have demonstrated that bone marrow mesenchymal stem cells (MSCs) are attractive candidates for cell and gene therapies, because they are readily obtained and multipotentially differentiated. Then homogeneous MSC cultures in vitro with more rapidly self-renewing ability and multipotential differentiation will accelerate and improve their progress in clinical application. Colter et al. found that early colonies contain a third kind of cells very small round cells that rapidly self-renew, besides spindle-shaped cells and large flat cells, called RS cells. RS cells are characterized by their extremely small size, rapid rate of replication, and enhanced potential for multilineage differentiation. Moreover, they can be distinguished from more mature cells in the same cultures by a series of surface epitopes and expressed proteins. Therefore, the results raise the possibility that RS cells may have the greatest potential for long-term engraftment and differentiation in vivo.
Bone Marrow Cells ; cytology ; Cell Differentiation ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; Multipotent Stem Cells ; classification ; cytology ; Stromal Cells ; cytology

The aim of this study was to isolate, cultivate and phenotypically characterize two types of human amnio-tic membrane (HAM)-derived cells, and to analyze their differentiation potential in vitro. Human amnion epithelial cells (hAEC) were derived from the embryonic ectoderm, while human amnion mesenchymal cells (hAMC) were derived from the embryonic mesoderm. The cells were characterized by flow cytometry and immunofluorescence, then immunofluorescence also was performed for the analysis of multipotentiality in differentiation. The results indicated that immunophenotypic characterization of both cell types demonstrated positive for HLA-A, B, C and mesenchymal stem cell markers (CD29, CD73, CD44, CD59, CD90, CD105, CD166), but did not express the hematopoietic markers (CD31, CD34, CD45, HLA-DR) and showed the weak expression of costimulatory molecules (CD40, CD40L, CD80, CD86). Phenotypes of both cell populations were maintained from passages 3 to 7. The immunofluorescence indicated that hAEC expressed cytokeratin 19, but did not express vimentin. On the contrary, hAMC expressed vimentin but did not express cytokeratin 19. The assessment of multilineage potential demonstrated that hAMC showed greater cardiomyocytes potential, while hAEC showed greater neural potential. It is concluded that hAEC and hAMC can be successfully isolated from the HAM. Both cell populations possess similar immunophenotype. However, they differ in cell yield and multipotential for differentiation into the major lineages, hAEC possess a much greater ectodermal differentiation capacity, while hAMC possess a much greater mesodermal differentiation capacity. This conclusion will be important for use of these cells in cell therapy.
Amnion ; cytology ; Cell Differentiation ; physiology ; Cell Lineage ; Epithelial Cells ; cytology ; Humans ; Immunophenotyping ; Stromal Cells ; cytology

OBJECTIVETo explore the mechanism of Wnt and Notch pathway involved modulating time and spatial restricted hematopoiesis.

METHODSMurine hematopoietic stem and progenitor cells (HSPCs) were isolated from bone marrow (BM) by using c-kit microbeads. E10.5 aorta-gonad-mesonephros (AGM), E12.5, E14.5, E16.5 fetal liver (FL) and adult BM derived stromal cells (StroCs) were isolated and co-cultured with c-kit(+)HSPCs. The floating cells in co-culture system were sorted and counted by FACS. Gene expressions of Wnt and Notch pathway were detected by quantitative PCR and protein expressions by immunostaining.

RESULTSCo-culturing HSPCs with AGM and FL-derived StroCs resulted in an expansion of c-kit(+)population from 0.4 x 10(5)/well to (19.2 +/- 3.2) x 10(5)/well and (26.8 +/- 5.4) x 10(5)/well, respectively, being greater than that with BM-derived StroCs (P < 0.05). The percentage of c-kit(+)cells detected in AGM- and BM- derived StroCs culture system was (75.2 +/- 7.1)%, (74.1 +/- 6.2)% respectively, being higher than FL- derived StroCs culture system (63.4 +/- 5.3)% (P < 0.05). Wnt and Notch pathway genes expression varied at different phases of hematopoiesis. Wnt was highly expressed in AGM and FL derived StroCs, and, Notch did in AGM and BM derived StroCs.

CONCLUSIONWnt and Notch pathway are important modulators in regulating time and spatial restricted hematopoiesis.

Animals ; Aorta ; cytology ; Coculture Techniques ; Hematopoiesis ; Hematopoietic Stem Cells ; cytology ; Humans ; Mesonephros ; cytology ; Stromal Cells

OBJECTIVETo investigate the biological function of hepatocyte-like cells derived from mesenchymal stem cells that isolated from human umbilical cord UC-MSCs in vitro, and to detect the changes in the immunogenicity of the differentiated hepatocyte-like cells (DHC).

METHODSTransdifferentiation of UC-MSCs into hepatic lineage in vitro was induced in modified two-step induction medium. The expressions of hepatic specific markers were detected by RT-PCR analysis and immunofluorescence staining at different time points after induction. The levels of albumin and urea in the supernatants of cultures were measured by enzyme-linked immunosorbent assay. Furthermore, the immunosuppressive property of DHC was detected by one-way mixed lymphocyte culture.

RESULTSThe mRNA and proteins of alpha fetoprotein (AFP), albumin (ALB),and cytokeratin-19 (CK-19) were expressed in naive UC-MSCs at low levels. DHC highly expressed hepatic markers AFP, ALB, CK-19, and tryptophan 2, 3-dioxygenase 14 and 28 days after hepatic differentiation and were accompanied by an increased production of ALB and urea in supernatant in a time-dependent manner. DHC did not express human leukocyte antigen DR antigen and significantly decreased the lymphocyte proliferation.

CONCLUSIONUC-MSCs are able to differentiate into functional hepatocyte-like cells in vitro, while the immunogenicity of DHC remains low.

Cell Transdifferentiation ; Cells, Cultured ; Hepatocytes ; cytology ; immunology ; Humans ; Mesenchymal Stromal Cells ; cytology ; Umbilical Cord ; cytology

OBJECTIVETo investigate the possibility of transdifferentiation of adipose mesenchymal stem cells (AMSCs) into hepatocytes.

METHODSHuman omentum adipose tissue was dispersed with collagenase I. Cells collected were cultured in a DMEM-F12 medium containing 2% FBS supplemented with 20 ng/ml HGF, 10 ng/ml FGF4, 1xITS and 0.1 micromol/L dexasmison. The cells of the control group were also cultured in the same kind of medium but without any cytokines serving as a control. The expression of hepatic transcriptional factors such as GATA4 and HNF1 were checked by RT-PCR. At the end of the induction, hepatocyte markers were analysed by flow cytometry, and cytokeratin expressions were examined using cyto-immunofluorescence methods.

RESULTSAMSCs grew like fibroblasts and were passaged easily. Most of the third passaged AMSCs were positive against anti-CD29, anti-CD44 antibodies, but negative for the anti-CD34 and anti-CD45 ones. The hepatic transcriptional factor was expressed gradually to higher levels during the induction time. AFP and Alb positive cells were 30.0% and 17.8% of the total cultured cells, and the rate of cells positive to the two markers was 6.9%. The inducted cells were positive for CK18 and CK19 antibodies at the end of the induction. The cells in the control group were negative when checked by these methods.

CONCLUSIONSAMSCs could be directed to differentiate into hepatocytes in vitro by a cytokine cocktail with a low concentration FBS culture system.

Adipocytes ; cytology ; Cell Differentiation ; Cell Transdifferentiation ; Cells, Cultured ; Hepatocytes ; cytology ; Humans ; Mesenchymal Stromal Cells ; cytology

Adult stem cells are drawing more and more attention due to the potential application in degenerative medicine without posing any moral problem. There is growing evidence showing that the human amnion contains various types of adult stem cell. Since amniotic tissue is readily available, it has the potential to be an important source of regenerative medicine material. In this study we tried to find multipotent adult stem cells in human amnion. We isolated stem cells from amniotic mesenchymal cells by limiting dilution assay. Similar to bone marrow derived mesenchymal stem cells, these cells displayed a fibroblast like appearance. They were positive for CD105, CD29, CD44, negative for haematopoietic (GlyA, CD31, CD34, CD45) and epithelial cell (pan-CK) markers. These stem cells had the potential to differentiate not only into osteogenic, adipogenic and endothelial lineages, but also hepatocyte-like cells and neural cells at the single-cell level depending on the culture conditions. They had the capacity for self-renewal and multilineage differentiation even after being expanded for more than 30 population doublings in vitro. So they may be an ideal stem cell source for inherited or degenerative diseases treatment.
Adult Stem Cells ; cytology ; Amnion ; cytology ; Cell Differentiation ; physiology ; Humans ; Mesenchymal Stromal Cells ; cytology ; Multipotent Stem Cells ; 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

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

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

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