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1.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(6): 1736-1741, 2019 Dec.
Artículo en Chino | MEDLINE | ID: mdl-31839031

RESUMEN

OBJECTIVE: To explore the role of bone marrow niche in the chemotherapy resistance of patient with acute myeloid leukemia (AML), and to investigate the effects of the MSCs on the apoptosis of HL-60 cell and its underlying mechanisms. METHODS: MSCs were derived from the bone marrow of newly diagnosed AML patients (AML-MSCs) and health donors(MSCs) were co-cultured with HL-60 cells respectively. The apoptosis of HL-60 cells in the presence/absence of MSCs and/or Daunorubicin were determined by flow cytometry with Annexin V/PI double staining. In addition, the morphological features of HL-60 cells were observed by Wright-Giemsa staining, and the ratio of blasts and differentiated cells were counted. Furthermore, the expressions of apoptosis-related factors including Caspase-3, Caspase-8,Caspase-9 and Survivin were detected by Western blot. RESULTS: The flow cytometry showed that there was no significant change in apoptosis of HL-60 cells co-cultured with MSC derived from healthy donors or AML patients. After adding Daunorubicin into different cultural systems, the apoptotic rates of HL-60, HL-60 co-cultured with normal MSCs and HL-60 co-cultured with AML-MSCs were (49.57±7.44)%, (30.72±4.05)% and (22.99±4.08)%, respectively, which showed that normal MSCs and AML-MSCs could remarkably supress Daunorubicin-induced HL-60 apoptosis, however, there was no statistically significant difference of apoptosis between HL-60 co-cultured with normal MSCs and HL-60 co-cultured with AML-MSCs. Wright-Giemsa staining showed that most of the HL-60 cells co-cultured with AML-MSCs were primitive, and cell differentiation was unusual. In AML-MSCs co-cultured group, the cell apoptosis and differentiation caused by DNR was significant decreased, and most of HL-60 cells were initial. Western blot showed that the cleavage activity of Caspase-3 of HL-60 in AML-MSCs and normal MSCs co-cultured group was decreased, compared with HL-60 in single cultured group, moreover, the decrease was significantly in AML-MSC group. Additionally, the expression of survivin in AML-MSCs and normal MSCs co-cultured group was increased, compared with that in single cultured group, and increase was significant in AML-MSCs group. CONCLUSION: MSCs can suppress Daunorubicin-induced HL-60 apoptosis via inhibiting Caspase-3 and maintaining survivin level.


Asunto(s)
Leucemia Mieloide Aguda , Células Madre Mesenquimatosas , Apoptosis , Células de la Médula Ósea , Caspasa 3 , Proliferación Celular , Daunorrubicina , Células HL-60 , Humanos , Survivin
2.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(4): 1259-1264, 2019 Aug.
Artículo en Chino | MEDLINE | ID: mdl-31418390

RESUMEN

OBJECTIVE: To explore the role of bone marrow microenvironment(niche) in the development of acute myeloid leukemia (AML) and the effect of AML patients-derived MSC on the proliferation, cell cycle and immuno-phenotypes of HL-60 cells. METHODS: The MSC derived from bone marrow of patients with newly diagnosed AML were isolated and co-cultured with HL-60 cells. The effect of MSC on proliferation of HL-60 cells was detected by using 3H-TdR incorporation method, the cell cycle and immunophenotypes of HL-60 cells were detected by flow cytometry. RESULTS: The results of 3H-TdR incorporation assay showed that both AML-MSCs and normal MSCs remarkably suppressed the HL-60 cell proliferation in a time- and dose-dependent manner. The results of cell cycle analysis demonstrated that AML MSCs and normal MSCs induced arrest of the HL-60 cells in G0/G1 phase. The results of immunophenotyping revealed that MSCs suppressed the expression of CD11a and CD154 on the surface of HL-60 cells. Moreover, AML MSCs exhibited increased inhibitory effects than that of normal MSCs. However, no remarkable effect of MSCs on CD54 expressions of HL-60 cells was observed in the current study. CONCLUSION: AML-MSCs possess effects on HL-60 cell proliferation, cell cycle and immunophenotypes similiar to normal MSCs, but exhibited increased suppressive capacity on the expression of CD11a and CD154.


Asunto(s)
Leucemia Mieloide Aguda , Células Madre Mesenquimatosas , Células de la Médula Ósea , Ciclo Celular , Proliferación Celular , Células HL-60 , Humanos , Inmunofenotipificación , Microambiente Tumoral
3.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(4): 1277-1286, 2019 Aug.
Artículo en Chino | MEDLINE | ID: mdl-31418393

RESUMEN

OBJECTIVE: To study the influence of acute myeloid leukemia (AML) microenvironment on mesenchymal stem cells (MSCs). METHODS: MSCs were isolated from the bone marrow of newly diagnosed AML patients (AML-MSCs) and were cultured. The morphology of MSC was observed by inverted microscopy, the immunophenotypes of MSC were detected by flow cytometry, the proliferation ability of MSC was detected by using MTT method, the multi-differentation ability of MSC was assayed by osteogenic, lipogenic and chrondrogenic induction. The morphologic features, immunophenotypic characteristics, cell proliferation, and multipotential differentiation capability were compared between the MSC derived from normal healthy donors and AML patients. RESULTS: AML-MSCs presented the morphological features similar to the normal MSCs. In addition, AML-MSCs highly expressed CD29, CD44, CD73, CD105 and HLA-ABC. Meanwhile, they were homogenously negative for CD14,CD31, CD34, CD45, CD80, CD86 and HLA-DR. Further-more, AML-MSCs showed cell proliferation ability similar to normal MSCs. Notably, AML-MSCs exerted increased osteogenic-differentiation capacity as compared with normal MSCs. CONCLUSION: AML-MSCs possess typical MSC phenotypes but displayed enhanced osteogenic-differentiation capacity.


Asunto(s)
Leucemia Mieloide Aguda , Células Madre Mesenquimatosas , Células de la Médula Ósea , Diferenciación Celular , Células Cultivadas , Humanos , Osteogénesis , Microambiente Tumoral
4.
Dev Dyn ; 240(1): 65-74, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21089075

RESUMEN

Mesenchymal stem cells (MSCs) represent powerful tools for regenerative medicine for their differentiation and migration capacity. However, ontogeny and migration of MSCs in mammalian mid-gestation conceptus is poorly understood. We identified canonical MSCs in the mouse embryonic day (E) 11.5 dorsal aorta (DA). They possessed homogenous immunophenotype (CD45(-)CD31(-)Flk-1(-)CD44(+)CD29(+)), expressed perivascular markers (α-SMA(+)NG2(+)PDGFRß(+)PDGFRα(+)), and had tri-lineage differentiation potential (osteoblasts, adipocytes, and chondrocytes). Of interest, MSCs were also detected in E12.5-E13.5 embryonic circulation, 24 hr later than in DA, suggesting migration like hematopoietic stem cells. Functionally, E12.5 embryonic blood could trigger efficient migration of DA-MSCs through platelet-derived growth factor (PDGF) receptor-, transforming growth factor-beta receptor-, but not basic fibroblast growth factor receptor-mediated signaling. Moreover, downstream JNK and AKT signaling pathway played important roles in embryonic blood- or PDGF-mediated migration of DA-derived MSCs. Taken together, these results revealed that clonal MSCs developed in the mouse DA. More importantly, the embryonic circulation, in addition to its conventional transporting roles, could modulate migration of MSC during early embryogenesis.


Asunto(s)
Aorta/embriología , Movimiento Celular/fisiología , Embrión de Mamíferos/irrigación sanguínea , Células Madre Mesenquimatosas/fisiología , Circulación Placentaria/fisiología , Animales , Aorta/citología , Aorta/fisiología , Diferenciación Celular , Linaje de la Célula/inmunología , Linaje de la Célula/fisiología , Células Cultivadas , Embrión de Mamíferos/citología , Femenino , Inmunofenotipificación , Ratones , Ratones Endogámicos C57BL , Embarazo , Células Madre/fisiología
5.
In Vivo ; 24(5): 659-66, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20952730

RESUMEN

BACKGROUND: Recent data have demonstrated that mesenchymal stem cells (MSCs) have potent immune regulation capacity in vitro, enhancing their therapeutic appeal for their utilisation in the management of acute graft-versus-host disease (aGvHD). However, their immunoregulatory activity in vivo is largely unknown. MATERIALS AND METHODS: Using murine compact bone-derived MSCs in an aGvHD model, the phenotypic status of splenocytes of aGvHD and aGvHD+MSC groups of mice were examined by flow cytometry. RESULTS: MSC infusion decreased the expression of MHC-II and CD69 molecules on splenic CD11b+ cells of aGvHD mice, which resulted in decreased maturation of antigen-presenting cells. Moreover, the down-regulated ratio of CD3+CD69+ to CD3+ cells, leading to restrained early activation and effector T-cell formation, resulted in the enhancement of the absolute and relative number of splenic CD3+ cells by MSCs co-transfer. CONCLUSION: This study demonstrated that MSCs can inhibit the three developmental stages of aGvHD.


Asunto(s)
Enfermedad Injerto contra Huésped/terapia , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/inmunología , Enfermedad Aguda , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Antígeno CD11b/metabolismo , Complejo CD3/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Enfermedad Injerto contra Huésped/inmunología , Inmunofenotipificación , Lectinas Tipo C/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Bazo/citología , Bazo/inmunología , Linfocitos T/inmunología
6.
Haematologica ; 95(6): 875-83, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20007140

RESUMEN

BACKGROUND: The hemangioblast is a bi-potential precursor cell with the capacity to differentiate into hematopoietic and vascular cells. In mouse E7.0-7.5 embryos, the hemangioblast can be identified by a clonal blast colony-forming cell (BL-CFC) assay or single cell OP9 co-culture. However, the ontogeny of the hemangioblast in mid-gestation embryos is poorly defined. DESIGN AND METHODS: The BL-CFC assay and the OP9 system were combined to illustrate the hemangioblast with lymphomyeloid and vascular potential in the mouse aorta-gonad-mesonephros region. The colony-forming assay, reverse transcriptase polymerase chain reaction analysis, immunostaining and flow cytometry were used to identify the hematopoietic potential, and Matrigel- or OP9-based methods were employed to evaluate endothelial progenitor activity. RESULTS: Functionally, the aorta-gonad-mesonephros-derived BL-CFC produced erythroid/myeloid progenitors, CD19(+) B lymphocytes, and CD3(+)TCRbeta(+) T lymphocytes. Meanwhile, the BL-CFC-derived adherent cells generated CD31(+) tube-like structures on OP9 stromal cells, validating the endothelial progenitor potential. The aorta-gonad-mesonephros-derived hemangioblast was greatly enriched in CD31(+), endomucin(+) and CD105(+) subpopulations, which collectively pinpoints the endothelial layer as the main location. Interestingly, the BL-CFC was not detected in yolk sac, placenta, fetal liver or embryonic circulation. Screening of candidate cytokines revealed that interleukin-3 was remarkable in expanding the BL-CFC in a dose-dependent manner through the JAK2/STAT5 and MAPK/ERK pathways. Neutralizing interleukin-3 in the aorta-gonad-mesonephros region resulted in reduced numbers of BL-CFC, indicating the physiological requirement for this cytokine. Both hematopoietic and endothelial differentiation potential were significantly increased in interleukin-3-treated BL-CFC, suggesting a persistent positive influence. Intriguingly, interleukin-3 markedly amplified primitive erythroid and macrophage precursors in E7.5 embryos. Quantitative polymerase chain reaction analysis demonstrated declined Flk-1 and elevated Scl and von Willebrand factor transcription upon interleukin-3 stimulation, indicating accelerated hemangiopoiesis. CONCLUSIONS: The hemangioblast with lymphomyeloid potential is one of the precursors of definitive hematopoiesis in the mouse aorta-gonad-mesonephros region. Interleukin-3 has a regulatory role with regards to both the number and capacity of the dual-potential hemangioblast.


Asunto(s)
Aorta/fisiología , Gónadas/fisiología , Hemangioblastos/fisiología , Interleucina-3/fisiología , Mesonefro/fisiología , Animales , Aorta/citología , Aorta/embriología , Células Cultivadas , Técnicas de Cocultivo , Gónadas/citología , Gónadas/embriología , Hemangioblastos/citología , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Humanos , Mesonefro/citología , Mesonefro/embriología , Ratones , Células del Estroma/citología , Células del Estroma/fisiología
7.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 17(4): 974-6, 2009 Aug.
Artículo en Chino | MEDLINE | ID: mdl-19698241

RESUMEN

This study was aimed to investigate the effect of activated T cell on the ability of MSC to differentiate into osteoblasts. The activated T cells with MSCs were co-culture for 14 days, then the osteoblast formation was tested by alkaline phosphatase staining. Furthermore, the supernatant of activated T cell was added in culture system of MSCs, the expression of molecules related with immune regulation of activated T cells was detected by RT-PCR, so as to determine what kinds of cytokine displayed the important function in MSC differentiation. The result showed that activated T cell could promote differentiation of MSC into osteoblasts, and IL-1beta played an important role in the effect of activated T cells on MSCs, while TNF-alpha, TGF-beta1 were not. It is concluded that the activated T cells promote the differentiation of MSCs to osteoblasts. The interactive influence between MSCs and immune cells can be mediated through cytokines.


Asunto(s)
Diferenciación Celular , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Linfocitos T/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Medios de Cultivo Condicionados , Humanos , Interleucina-1beta/biosíntesis
8.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 17(4): 977-80, 2009 Aug.
Artículo en Chino | MEDLINE | ID: mdl-19698242

RESUMEN

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.


Asunto(s)
Artritis Reumatoide , Células de la Médula Ósea/citología , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Líquido Sinovial/citología , Diferenciación Celular , Células Cultivadas , Humanos
9.
Stem Cells Dev ; 18(10): 1473-84, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19374589

RESUMEN

Mesenchymal stem cells (MSCs) are characterized by their hematopoiesis-supporting and immunosuppressive capacity, while osteoclasts are main cell components in the endosteal hematopoietic stem cell niche and pivotal players in osteoimmunology. To clarify the association of these 2 kinds of cells, mouse CD11b(+) monocytes were cultured onto MSC layers in the presence or absence of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). The results showed that MSCs independently supported osteoclast development and this effect was enhanced by M-CSF and RANKL. Interestingly, tumor necrosis factor-alpha (TNF-alpha)-stimulated MSCs turned to inhibit osteoclast formation and protect tusk slices from osteoclastic resorption. Real-time PCR and ELISA assays demonstrated that osteoprotegerin expression at both mRNA and protein levels in TNF-alpha-stimulated MSCs was up-regulated, at least partially by activating the mitogen-activated protein kinase pathway. Furthermore, TNF-alpha-stimulated MSCs maintained their immunophenotypic, multipotential differentiation and immunosuppressive characteristics. Moreover, MSCs treated with synovial fluid from rheumatoid arthritis patients modulated osteoclast generation in close relation with the TNF-alpha levels. This study suggests that MSCs exhibit dual modulatory function on osteoclasts and the result might shed light on understanding the involvement of MSCs in the inflammatory diseases.


Asunto(s)
Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Animales , Artritis Reumatoide/patología , Antígeno CD11b/metabolismo , Diferenciación Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Inmunofenotipificación , Masculino , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Células Madre Multipotentes/citología , Células Madre Multipotentes/efectos de los fármacos , Células Madre Multipotentes/metabolismo , Osteoclastos/enzimología , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Solubilidad/efectos de los fármacos , Líquido Sinovial/citología , Líquido Sinovial/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos
10.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 15(5): 1019-22, 2007 Oct.
Artículo en Chino | MEDLINE | ID: mdl-17956682

RESUMEN

This study was aimed to investigate whether mesenchymal stem cells (MSCs) existed in human umbilical cord vein and to establish the methods of isolation and expansion in vitro. The MSCs derived for perfusion of umbilical cord vein (UVMSCs) were collected after parturition of Healthy pregnant women. The morphology of MSCs and their differentiation potential into osteoblast, adipocyte and chondrocyte were observed the phenotype and cell cycle of MSCs were determined by using flow cytometry. The result showed that the mesenchymal stem cells separated from umbilical cord vein gave rise to a population of adherent cells with a typical fibroblast-like morphology. Similarly to bone marrow-derived MSCs, they highly expressed CD29, HLA-ABC, CD166, CD105, CD73 and CD44, and were negative for any hematopoietic and endothelial markers (CD45, CD34, CD14 and CD144). Functionally, they could differentiate into the osteoblast, adipocyte and chondrocyte. It is concluded that MSCs exist in the human umbilical cord vein perfusion. Their read amplification in vitro contribute to clinical applications for cell therapy and tissue engineering.


Asunto(s)
Separación Celular/métodos , Sangre Fetal/citología , Células Madre Mesenquimatosas/citología , Venas Umbilicales/citología , Diferenciación Celular , Células Cultivadas , Femenino , Humanos
11.
Cell Transplant ; 16(1): 85-95, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17436858

RESUMEN

Previous data have demonstrated that mesenchymal stem cells (MSCs) can exert immunomodulatory activity in vitro, in which of the process nearly all kinds of immune cell subsets are involved. However, there is still a paucity of information about whether and why MSCs inhibit the ongoing immune responses in vivo. Working in a murine splenocyte transfusion model across the major histocompatibility barrier (C57BL/6 -BALB/c, H2b --> H2d), we have found that MSC coinfusion prolongs the mean survival time (MST) of the recipient mice in a dose-dependent manner and reduces graft-versus-host-associated histopathology in comparison to the allosplenocyte transfusion controls. In vivo eGFP tracing with polymerase chain reaction analysis revealed that grafted MSCs could migrate and settle into the lungs, spleen, liver, intestine, and skin shortly after administration. Further investigations into the functional characteristics of intrasplenic lymphocytes showed that their proliferation and cytotoxic activity against P815 cells (H2d) were significantly restrained by MSC cotransfer. FACS analysis demonstrated that MSC infusion not only increased the proportion of CD4+ subset but also decreased that of CD8+ cells at the belated observation points, resulting in the increase of the ratio of CD4+/CD8+ cells. Also, in contrast to the slight increase of the proportion of CD4+CD25+ T regulatory cells (Tregs) in MSC cotransfer mice, the ratio of Tregs/CD8+ cells was dramatically elevated. Furthermore, RT-PCR analysis on the cytokine array of IL-2, IL-4, IL-12, TNF-alpha, and TGF-beta in recipient splenocytes implied the Thl to Th2 polarization. Therefore, it is deducible that alteration in the proportions of different T-lymphocyte subsets may be one of the main mechanisms by which grafted MSCs suppress the ongoing immune responses in vivo. The study here might provide some new clues for the design of therapeutic approaches for MSC transplantation.


Asunto(s)
Trasplante de Células Madre Mesenquimatosas , Bazo/inmunología , Linfocitos T/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Proliferación Celular , Citocinas/metabolismo , Femenino , Transfusión de Linfocitos , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fenotipo , Bazo/citología , Bazo/trasplante , Linfocitos T/citología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo
12.
Cell Transplant ; 16(1): 85-95, 2007 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-28880677

RESUMEN

Previous data have demonstrated that mesenchymal stem cells (MSCs) can exert immunomodulatory activity in vitro, in which of the process nearly all kinds of immune cell subsets are involved. However, there is still a paucity of information about whether and why MSCs inhibit the ongoing immune responses in vivo. Working in a murine splenocyte transfusion model across the major histocompatibility barrier (C57BL/6 → BALB/c, H2b → H2d), we have found that MSC coinfusion prolongs the mean survival time (MST) of the recipient mice in a dose-dependent manner and reduces graft-versus-host-associated histopathology in comparison to the allosplenocyte transfusion controls. In vivo eGFP tracing with polymerase chain reaction analysis revealed that grafted MSCs could migrate and settle into the lungs, spleen, liver, intestine, and skin shortly after administration. Further investigations into the functional characteristics of intrasplenic lymphocytes showed that their proliferation and cytotoxic activity against P815 cells (H2d) were significantly restrained by MSC cotransfer. FACS analysis demonstrated that MSC infusion not only increased the proportion of CD4+ subset but also decreased that of CD8+ cells at the belated observation points, resulting in the increase of the ratio of CD4+/CD8+ cells. Also, in contrast to the slight increase of the proportion of CD4+CD25+ T regulatory cells (Tregs) in MSC cotransfer mice, the ratio of Tregs/CD8+ cells was dramatically elevated. Furthermore, RT-PCR analysis on the cytokine array of IL-2, IL-4, IL-12, TNF-α, and TGF-ß in recipient splenocytes implied the Th1 to Th2 polarization. Therefore, it is deducible that alteration in the proportions of different T-lymphocyte subsets may be one of the main mechanisms by which grafted MSCs suppress the ongoing immune responses in vivo. The study here might provide some new clues for the design of therapeutic approaches for MSC transplantation.

13.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 14(6): 1191-4, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17204192

RESUMEN

This study was aimed to investigate if human heart harbored a population of primitive undifferentiated cells with the characteristics of MPC. Cells were isolated from human fetal heart and were cultured under conditions appropriate for bone marrow-derived MPCs. Their morphology, phenotypes and functions were tested by methods developed for MPC from other sources. The results showed that morphologically, cells were spindle shaped and resembled fibroblasts. In their undifferentiated state, cells were CD73, CD105, CD29, CD44, HLA-ABC, CD166 positive and CD45, CD34, CD86, HLA-DR negative. When cultured in adipogenic, osteogenic or chondrogenic media, cells differentiated into adipocytes, osteocytes and chondrocytes respectively. They could be extensively expanded in vitro and exhibited very low immunogenicity as evaluated by T cell proliferation assays. It is concluded that cells isolated from fetal heart possess similarity to their adult and fetal bone marrow counterparts in morphologic, immunophenotypic, and functional characteristics.


Asunto(s)
Células de la Médula Ósea/citología , Corazón Fetal/citología , Células Madre Mesenquimatosas/citología , Adhesión Celular , Diferenciación Celular , Células Cultivadas , Feto , Humanos , Células Madre Mesenquimatosas/inmunología , Células Madre Multipotentes/fisiología
14.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 13(1): 50-3, 2005 Feb.
Artículo en Chino | MEDLINE | ID: mdl-15748435

RESUMEN

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.


Asunto(s)
Células de la Médula Ósea/citología , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Células de la Médula Ósea/inmunología , Diferenciación Celular/inmunología , Linaje de la Célula , Proliferación Celular , Células Cultivadas , Humanos , Células Madre Mesenquimatosas/inmunología , Osteoblastos/inmunología , Linfocitos T/citología , Linfocitos T/inmunología
15.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 11(5): 530-3, 2003 Oct.
Artículo en Chino | MEDLINE | ID: mdl-14575553

RESUMEN

The study was aimed to evaluate if the modified in situ cryopreservation could affect the biological function of mesenchymal stem cells (MSC) in vitro. Mesenchymal stem cells from human bone marrow were isolated by standard method and characterized with their morphology, cell-surface antigen profile and differentiation repertoire in vitro. The culture-expanded MSC were cryopreserved in situ with culture medium (DMEM-LG) containing 10% D MSO and 30% selected FCS in -70 degrees C. Following recovery of cryopreservation, differentiation to adipocytes, chondrocytes, and osteoblast in vitro and cell cycle analysis were performed to investigate whether the cryopreservation would change the differentiation potential of MSC. The results showed that after recovery of cryopreservation, there was no changes detected as compared with the culture-expanded MSC in both differentiation potency and growth pattern at 12 weeks. In conclusions: this optimized short term in situ cryopreservation at -70 degrees C could retain biological characteristics of human MSC for at least 3 months, and this method may be useful for cryopreservation of hum an bone marrow MSCs.


Asunto(s)
Células de la Médula Ósea/citología , Criopreservación , Células Madre Mesenquimatosas/citología , Ciclo Celular , Diferenciación Celular , Separación Celular , Supervivencia Celular , Humanos
16.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 10(2): 112-4, 2002 Apr.
Artículo en Chino | MEDLINE | ID: mdl-12513810

RESUMEN

In order to study the relationship between the expression of glutathione S-transferase (GST) in leukemic cells and the chemoresistance in patients with acute leukemia, the expressions of GST activity and GST mRNA were measured according to spectrophotometric assay based on the use of 1-choloro-2, 4-dinitro benzene and in situ hybridization. The results were studied in correlation with some clinical and pathological data. Results showed that: 1. There is no significant differences between activities of the enzyme with the different leukemia types according to the FAB classification. 2. GST activity and GST mRNA expression in the patients, both untreated and relapse, were (4.5 +/- 1.0) U, 33.3% and (7.9 +/- 15) U, 66.3% respectively. 3. In 56 patients, GST activity was 1.7 +/- 0.7, 5.9 +/- 2.0 and 9.3 +/- 1.7 U and GST mRNA expression was 13.3%, 29.7% and 76.6%, respectively, in CR, PR and NR groups. The lowest values of GST activity and GST mRNA expression were observed in those patients who achieved complete remission. The highest values of GST activity and GST mRNA expression were observed in those patients with no response to treatment. It was concluded that the expression of GST in patients with acute leukemia is closely related to the chemosensitivities clinically. Determinations of GST activity and GST mRNA are useful for predicting the chemosensitivities and the prognosis of the disease.


Asunto(s)
Glutatión Transferasa/metabolismo , Leucemia/enzimología , Adolescente , Adulto , Anciano , Resistencia a Antineoplásicos , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glutatión Transferasa/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Células K562 , Leucemia/tratamiento farmacológico , Leucemia/genética , Leucemia Linfoide/tratamiento farmacológico , Leucemia Linfoide/enzimología , Leucemia Linfoide/genética , Leucemia Monocítica Aguda/tratamiento farmacológico , Leucemia Monocítica Aguda/enzimología , Leucemia Monocítica Aguda/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/enzimología , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo
17.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 9(3): 212-214, 2001 Sep.
Artículo en Chino | MEDLINE | ID: mdl-12578591

RESUMEN

CD40/CD40L interactions play a pivotal role in T cell activation, and take part in many physiologic and pathologic procedures and different levels. In this article, stable CHO transformants secreting human CD40-Ig fusion protein were established through transfection and selection with Lipofectamaine and G418, respectively. In order to obtain great valume of recombinant protein, big batch serum-free cultures of engineered CHO cells were performed in roller-bottle using CHO-II-SFM medium. After cultures, the cell-culture supernatants were harvested, concentrated through ultra-filtration, and finally purified by affinity choromatography with Protein G Sepharose Fast Flow. Human peripheral bloods were collected freshly and seperated with Ficoll, CFU-T was cultured in semi-solid culture system with peripheral blood mononuclear cells (PBMNC). Effect of human CD40-Ig fusion protein on the formation of CFU-T was observed in vitro. The results showed that the yield of human CD40-Ig fusion protein was 30 mg in total 3 liter CHO-II-SFM culture supernatant, and it supposed that the expression level of CD40-Ig in CHO cells was more than 10 micro g/ml. The purity of purified fusion protein is above 95%. Furthermore, compared with human IgG, human CD40-Ig fusion protein significantly inhibited the formation of CFU-T at dose 0.25, 1.0, 4.0, and 10 micro g/ml, it lays a good foundation to evaluate its potential functions in vivo.

18.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 9(2): 97-100, 2001 Jun.
Artículo en Chino | MEDLINE | ID: mdl-12578611

RESUMEN

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.

19.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 9(1): 91-92, 2001 Mar.
Artículo en Chino | MEDLINE | ID: mdl-12578655

RESUMEN

Recent reports have clearly demonstrated that bone marrow cells can be differentiated into neurons, suggesting the existence of cells with the differentiation capacity in the bone marrow cell population. It is well known that hematopoietic stem cells as well as mesenchymal stem cells (MSCs) can be transplanted and therefore, alternative of them might contribute to the process. In the present study it was addressed whether marrow MSCs could be coaxed into neuron-specific antigen bearing cells and if so, whether the differentiated cells possess the cytochemical features seen in neurons. The report here showed that high concentration of 2-mercaptoethanol (2-ME) could induce some of the MSCs into neuron-like cells expressing neurofilament (NF) and neuron specific enolase (NSE). The neuron-like cells were alkaline phosphotase positive while the others MSCs were kept negative. Cells treated with 2-ME were positive for alpha-naphthylacetate esterase and glycogen and negative for acetylchonlinesterase, which were similar with the results seen in untreated cells. Furthermore, Nissel body was not observed in treated cells shown by toluidine blue staining. Therefore, it is likely that the cells described here seem not belong to the neuronal lineage. These findings, however, reveal that human MSCs could alter their committed fates under some circumstances.

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