This study was aimed to investigate whether the thrombopoietin (rhTPO) may facilitate myelofibrosis or not. The modified Dexter culture system with various concentrations of rhTPO was used to culture the stromal cells in vitro; the proliferative activity of cells was detected by MTT method; the morphologic changes were observed by light and scanning electron microscopy; the staining changes of ALP, PAS, AS-D NCE and IV type collagen were observed by cytochemistry method; the changes of fibronectin, laminin and IV type collagen were assayed by immunohistochemistry method; the cell surface antigens were assayed by flow cytometry. The results indicated that rhTPO could promote the proliferation of stromal cells which was related to the concentrations of rhTPO. Proliferative activity of stromal cells increased with increasing of rhTPO concentration, and was not related to the exposure time. On day 3 stromal cells adhered to the wall, and became oval. On day 7 stromal cells turned to fusiform and scattered dispersively. On day 12 to 14 these cells ranged cyclically and became long fusiform. Cells covered 70%-80% area of bottle bottom at that time. By day 16 to 18 these cells covered more than 90% area of bottom and ranged cyclically. They displayed the same shape as fibroblasts. By light microscopy with Wrights-Giemsa staining, fibroblasts predominated morphologically, few macrophages, endothelial cells and adipose cells were found. There were no significant differences between experimental group and control group. On day 14 to 42 the adherent cells were positive with PAS staining, poorly positive with ALP and naphthol AS-D chloroacetate esterase (AS-D NCE) staining, and the difference in cytochemistry was not significant between two groups. When these cells were dyed with Masson's trichrome and Gomori's staining, neither collagen fibers nor reticular fibers were positive, but fibronectin, laminin, and collagen type IV appeared positive stronger in experimental group than those in control. The expressions of these molecules were not dependent on culture time. By scanning electron microscopy microvilli and fibers on cell surface appeared more and more, monolayer cells evolved into multilayer cells, and newly-formed fibroblasts appeared gradually as culture time prolonged. These alterations were not different among various groups. The expressions of CD34, CD45, CD105, CD106, and CD166 were not affected obviously by rhTPO. It is concluded that rhTPO had no effects on histochemical properties of stromal cells. Fiber staining and scanning electron microscopic examinations revealed that rhTPO can not facilitate fiber formation of stromal cells. But rhTPO may be able to augment the expressions of fibronectin, laminin and collagen type IV of stromal cells. Therefore it is still necessary to follow up the patients for a long time, who have received rhTPO therapy clinically.
Bone Marrow Cells ; cytology ; drug effects ; Cell Proliferation ; drug effects ; Fibroblasts ; Humans ; Stromal Cells ; cytology ; drug effects ; Thrombopoietin ; pharmacology

Bone marrow stromal cells (BMSCs), a kind of stem cells residing in bone marrow, have self-renewal, high proliferative capacity and the potential of multilineage differentiation. It has a good prospect in application of the cell replacement therapy, the gene therapy and the tissue engineering and so on. As the content of BMSCs is extremely low in bone marrow, BM-SCs must be amplified in vitro and induced to differentiation to meet the clinical needs. Researches of the recent years suggest there is a very promising way that Chinese medicine could induce BMSCs proliferation, differentiation. Based on the Chinese medicine theory, "the kidney generating marrow and dominating bone" and "kidney storing essence, essence and marrow", the TCM scholars have done some researches to explore the function of warming yang and reinforcing kidney of Chinese medicine to promote bone marrow stromal cells and found that these drugs can promote the BMSCs to proliferate and to differentiate into osteogenic, cartilage and nerve cells. This article elaborates and presents the researches on this aspect.
Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; drug effects ; Humans ; Medicine, Chinese Traditional ; Stromal Cells ; cytology

Hypoxia in bone marrow is suitable for the perfect preservation of biological functions of bone marrow hematopoietic stem cells (BM HSC). It is deserved to study whether the biological functions of BM HSC are influenced when being exposed to environment of oxygen at various concentration during amplification of BM HSCs in normal oxygen condition in vitro and process of peripheral blood hematopoietic stem cell transplantation (PBSCT). This study was purposed to investigate the effects of various oxygen concentrations on biological functions of human BM HSCs. The BM HSCs were amplified in vitro, the amplification level of CD34(+) HSCs and CD34(+)AC133(+) HSCs were detected by flow cytometry, the apoptosis and cell cycle distribution of CD34(+) HSCs amplified in various oxygen concentrations were assayed by flow cytometry with Annexin V/PI double staining as well as PI and Ki-67 antibody, respectively, the differentiation of amplified CD34(+) HSCs in vitro was determined by direction differentiation assay, the migration ability of amplified CD34(+)AC133(+) HSCs was measured by migration test. The results indicated that the oxygen environment below normal oxygen, especially hypoxia, could amplify more primitive CD34(+)AC133(+) HSCs and CD34(+) HSCs with activity, arrest more HSCs in G₀/G₁ phase, promote the generation of BFU-E, CFU-GM, CFU-GEMM, and better preserve the migration ability of HSCs. While the above functional indicators of BM HSCs were poor when HSCs exposed to normoxia, oxygen-unstable and oxygen-severe changeable environments. It is concluded that the biological functions of BM HSCs in PBSCT are related with oxygen concentration and its stability, the culture of BM HSCs in lower oxygen environment may be more beneficial for PBSCT.
Bone Marrow Cells ; cytology ; drug effects ; Bone Marrow Transplantation ; Cell Hypoxia ; Cells, Cultured ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Oxygen ; administration & dosage ; pharmacology

Bone marrow mesenchymal stem cells (MSCs) have active abilities of self-replication and multidifferentiation. In recent years, a lot of studies have proved that MSCs can be induced and differentiated into nerve-like cells under certain conditions. Because of some advaced characteristics including sampling convenience, no immune rejection, high transfection rate and stable exogenous gene expression, MSCs will provide new way in treating disease of nervous system. In this article, the research progress of bone marrow mesenchymal stem cells differentiation into nerve-like cells induced by Traditional Chinese Medicine shall be discussed, and explore the research thinking guided by basis theory of TCM.
Animals ; Bone Marrow Cells ; cytology ; drug effects ; Cell Differentiation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Neurons ; cytology ; drug effects

To study the cartilage differentiation of mouse mesenchymal stem cells (MSCs) induced by cartilage-derived morphogenetic proteins-2 in vitro, the MSCs were isolated from mouse bone marrow and cultured in vitro. The cells in passage 3 were induced into chondrogenic differentiation with different concentrations of recombinant human cartilage-derived morphogenetic proteins-2 (0, 10, 20, 50 and 100 ng/mL). After 14 days of induction, morphology of cells was observed under phase-contrast microscope. Collagen II mRNA and protein were examined with RT-PCR, Western blotting and immunocytochemistry respectively and the sulfate glycosaminoglycan was measured by Alcian blue staining. RT-PCR showed that CDMP-2 could promote expression of collagen II mRNA in an dose-dependant manner, especially at the concentration of 50 ng/mL and 100 ng/mL. Immunocytochemistry and Western blotting revealed a similar change. Alcian blue staining exhibited deposition of typical cartilage extracellular matrix. Our results suggest that mouse bone marrow mesenchymal stem cells can differentiate into chondrogenic phonotype with the induction of CDMP-2 in vitro, which provides a basis for further research on the role of CDMP-2 in chondrogenesis.
Bone Marrow Cells/*cytology ; Bone Morphogenetic Proteins/*pharmacology ; Cell Differentiation/*drug effects ; Cells, Cultured ; Chondrocytes/*cytology ; Chondrogenesis/drug effects ; Chondrogenesis/physiology ; Mesenchymal Stem Cells/*cytology ; Recombinant Proteins/pharmacology

OBJECTIVETo investigate the role of mesenchymal stem cells (BMSCs) in multiple myeloma (MM) bone marrow (BM) microenrivonment and their effect on myeloma cells survival and bortezomib induced apoptosis.

METHODSBMSCs were derived from BM of untreated myeloma patients (MM-BMSCs) and healthy donors (HD-BMSCs), respectively. The phenotype, proliferation time and cytokine secretion of MM-BMSCs were detected and compared with HD-BMSCs. Then BMSCs were co-cultured with myeloma cell line NCI-H929 and bortezomib in vitro. The NCI-H929 cells proliferation and bortezomib induced cell apoptosis were investigated.

RESULTSMM-BMSCs and HD-BMSCs were isolated successfully. The phenotype of MM-BMSCs was similar to that of HD-BMSCs. Expressions of CD73, CD105, CD44 and CD29 were positive, but those of CD31, CD34, CD45 and HLA-DR (< 1%) negative. The proliferation time of MM-BMSCs was longer than that of HD-BMSCs (82 h vs 62 h, P < 0.05). Moreover, over-expressions of IL-6 and VEGF in MM-BMSCs culture supernatant were detected as compared with that in HD-BMSCs [(188.8 ± 9.4) pg/ml vs (115.0 ± 15.1) pg/ml and (1497.2 ± 39.7) pg/ml vs (1329.0 ± 21.1) pg/ml, respectively]. MM- BMSCs supported survival of the myeloma cells NCI-H929 and protected them from bortezomib induced cell apoptosis.

CONCLUSIONSMM-BMSCs is benefit for myeloma cells proliferation and against cell apoptosis induced by bortezomib. Over-expression of IL-6 and VEGF maybe play a critical role in these effects.

Apoptosis ; drug effects ; Bone Marrow Cells ; cytology ; Bortezomib ; Humans ; Mesenchymal Stromal Cells ; metabolism ; Multiple Myeloma ; metabolism

OBJECTIVETo establish an immortalized marrow mesenchymal stem cell line to facilitate advances in cartilage engineering research.

METHODSHuman telomerase reverse transcriptase (hTERT) cDNA was transferred into primary human marrow mesenchymal stem cells (hMSC) by retroviral vector pLEGFP-C1-hTERT. Subsequently G418 resistant cell clone was screened and expanded for further studies. hMSC biomarkers and hTERT expression were confirmed by examination. Transfected hMSC was induced to differentiate into chondrocyte using TGF-P1 and dexamethasone.

RESULTSUp-regulated hTERT expression was detected in transfected hMSC. hMSC-hTERT cells could be induced to differentiate into chondrocyte. Higher telomerase activity in transfected cells was maintained for 50 population doublings so far. Collagen II could be detected in induced transfected hMSC by immunocytochemical and hybridization in situ.

CONCLUSIONSEctopic expression of hTERT can effectively immortalize hMSC in vitro. Immortalized hMSC can be induced to differentiate into chondrocyte under certain condition. It may be an ideal target of further studies in cartilage engineering.

Bone Marrow Cells ; cytology ; drug effects ; metabolism ; Cell Differentiation ; drug effects ; Cell Line, Transformed ; Chondrocytes ; cytology ; Humans ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Telomerase ; genetics ; metabolism ; Transfection

Bone marrow mesenchymal stem cells (BMSCs) are a kind of pluripotent stem cells derived from bone marrows, which can not only support hematopoiesis, but also have capabilities of multidifferentiation, high-proliferation and self-renewing. They have become one of hotspots in stem cell studies. Studies on in vitro intervention with BMSCs with TCMs have made remarkable progress in recent years. According to the findings, some traditional Chinese medicines can promote proliferation of BMSCs, some can inhibit the apoptosis of BMSCs, while others can induce BMSCs to differentiate into multiple cell types, such as osteoblast. Furthermore, some studies also involved relevant action mechanisms. The authors summarized the advance in relevant studies by reference to relevant literatures of this field.
Animals ; Apoptosis ; drug effects ; Bone Marrow Cells ; cytology ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Humans ; Medicine, Chinese Traditional ; methods ; Mesenchymal Stromal Cells ; cytology ; drug effects

OBJECTIVETo explore the effects of fibrinogen(FG) and laminin(LN) in promoting the osteogenic differentiation of bone mesenchymal stem cells(BMSCs)in PEGDA scaffold.

METHODSAfter the rabbit BMSCs were isolated and cultured to passage 3. BMSCs were blended in PEGDA-FG or PEGDA-LN hydrogels and cultured for 7 days. The levels of osterix,osteopontin,osteocalcin,collagen 2,myocardin,PPARΓ,and integrins Α2,Α5,and Α6 in PEGDA-FG and PEGDA-LN constructs were determined. Immunohistochemistry was used to detect the expressions of myocardin,PPARΓ,and OPN in PEGDA-FG and PEGDA-LN constructs.

RESULTSThe expressions of osterix,OPN,and OC were significantly higher in PEGDA-FG scaffold than day 0(all P<0.05). The OPN and OC expression levels were significantly higher in PEGDA-LN scaffold than day 0(both P<0.05). In PEGDA-FG and PEGDA-LN scaffold,myocardin,PPARΓ and COL 2 expression level showed no significant differences than day 0(all P>0.05). Integrin Α2 was upregulated in PEGDA-LN scaffold than day 0(P<0.05). Integrin Α6 was upregulated in PEGDA-FG scaffold than day 0(P<0.05). Immunohistochemistry stain showed that OPN expression increased in PEGDA-FG and PEGDA-LN scaffolds.

CONCLUSIONFG and LN can promote rabbit BMSCs osteogenic differentiation in PEGDA three-dimensional scaffold.

Animals ; Bone Marrow Cells ; cytology ; drug effects ; Cell Differentiation ; Cells, Cultured ; Fibrinogen ; pharmacology ; Laminin ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Osteogenesis ; drug effects ; Rabbits ; Tissue Scaffolds

The purpose of this study was to investigate the effect of salidroside on human bone marrow mesenchymal stem cell (hBMMSC) apoptosis induced by cytarabine C (Ara-C) and its mechanism, hBMMSC were cultured in vitro and isolated by Fircoll density gradient centrifugation; cell surface antigens were measured by flow cytometry; the osteogenic and adipogenic differentiation of MSC was tested and evaluated by specific staining methods. The proliferation and apoptosis of cells exposed to Ara- C were detected by MTT and flow cytometry respectively. The experiments were divided into 4 groups: control group, Ara-C group, salidroside group and Ara-C+salidroside group. The mRNA expression of BCL-2 and BAX was assayed by RT-PCR. The results showed that the adherent cells displayed spindle and fibroblast cell-like shape; the hBMMSC expressed CD44, CD71 and HLA-ABC, not expressed CD34, CD45 and HLA-DR; the hBMMSC successfully differentiated into osteogenic and adipogenic lineages, which showed mineralization with von Kossa staining. Furthermore, liquid vacuoles were detected by oil red O staining; Ara- C exhibited a less inhibitory effect on the proliferation of hBMMSC treated with salidroside. The apoptosis of hBMMSC treated with salidroside were significantly higher as compared with control group (P < 0.05); RT-PCR results demonstrated that the BCL-2 expression was significantly down regulated but BAX mRNA expressions was up-regulated in Ara- C group as compared with those in the control group. Salidroside significantly inhibited the apoptosis of MSC and reversed the mRNA expression of BCL-2 and BAX. It is concluded that salidroside can inhibit the apoptosis of hBMMSC induced by Ara-C, its mechanism may be related with the regulation of BCL-2/BAX expression.
Apoptosis ; drug effects ; Bone Marrow Cells ; cytology ; drug effects ; Cells, Cultured ; Cytarabine ; pharmacology ; Glucosides ; pharmacology ; Humans ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Phenols ; pharmacology