| Objective To establish the methods of isolation and expansion of (human umbilical cord derived mesenchymal stem cell, hUC-MSC). By studying the basic biological characteristics of hUC-MSC and its effects on the homing, engraftment, reconstrution of hematopoiesis and its immunomodulatory function after cotransplantation with the hematopoietic stem/progenitor cells to confirm its capacity of hematpoietic-support which should open the horizons for hUC-MSC cotransplantation with hematopoietic stem/progenitor cells to improve the clinical outcome of transplatantions.Methods The study was divided into four parts: 1. hUC-MSCs were isolated and expanded from human umbilical cord after enzyme digested. The basic biological characteristics of hUC-MSC were investigated by the morphology investigation, growth curve drawing of proliferation and passage cultures, unit-fibroblast like colony-forming (CFU-F) assay. Cell cycle analysis and immunophenotype were also detected by flow cytometre (FCM). 2. RT-PCR technique was used to detect the gene expression of hematopoietic growth factors, chemokine and adhesion molecules associated with homing and engraftment of hUC-MSC. Colony-forming assay of hematopoietic stem/progenitor cell was also performed to test the capability of hUC-MSC to maintain hematpoiesis. A cell tracing technique was used to detect the spatial localization of transplanted hUC-MSCs in nonobese diabetic/severe combined immundeficicency (NOD/SCID) mouse afer 24h transplantation. The homing of human hematopoietic cells mediated by hUC-MSC after 24h cotransplantation was also detected through tracing technique. 3. In order to study the immunomodulatory capacity of hUC-MSC, FCM technique was used to detect the CD69 expression, which serves as an ideal indicator of a T-cell early activation, on PHA+PMA activated T cells after 8 hours hUC-MSCs coculture. The effect of hUC-MSC on T-lymphocyte proliferation induced by PHA were also detected by MTT. 4. Lastly a NOD/SCID mouse model of hUC-MSC cotransplantation with human umbilical cord blood was developed to detect the survival rate of NOD/SCID mouse after cotransplantation and the extent of engraftment of umbilical cord blood cells (chimerism of human CD45+ cells) in the bone marrow of NOD/SCID mouse were also measured by FCM.Results The primary hUC-MSCs isolated and cultured by such method adhered to culture flask within 24-48h. Unit-fibroblast like colony-forming (CFU-F) can be seen after 6-10d, and about 459±76 mononuclear cells digested from umbilical cord can form a fibroblast-like colone. After a 2-3 weeks primary expansion, hUC-MSCs can be passaged. The passaged hUC-MSCs proliferated quite rapidly and the number of hUC-MSCs in logarithemic growth period can be doubled within 32h. After 29d sequential passages about 9.82±0.79)×109 hUC-MSCs can be hearvested. Cell cycle analysis of hUC-MSCs showed more than 80 % cells were in G0-G1 phase. The immunophenotypic analysis showed that hUC-MSCs express CD13, CD29, CD44 and CD105, but negative expression of CD34, CD38 and CD45. The MHC phenotypic analysis indicated negative expression of HLA class II and negative or less expression of HLA class I. RT-PCR showed hUC-MSCs express hematopoietic growth factors such as SCF, Flt3L, IL-3, IL-6, G-CSF, GM-CSF, and also express chemokine receptor CXCR-4 and its ligend, stromal derived factor (SDF-1) and other adhesion molecules, CD11a/ICAM-1, CD49d /VCAM-1 associated with homing and engrftment. Colony-forming assay of hematopoietic stem/progenitor cell showed that hUC-MSC can maintain capability of hematpoietic colony-forming more than 6 weeks. Tracing technique showed the spatial localization of transplanted hUC-MSCs in NOD/SCID mouse afer 24h transplantation were mainly in marrow , less in liver and spleen, but no in peripheral blood, lung, kidney, heart and brain. The data showed that cotransplantation of hUC-MSCs together with umbilical cord blood (UCB) resulted in a significant increse of human UCB mononuclear cells (MNCs) in NOD/SCID mouse bone marrow nucleated cells as compared with the infusion of UCB alone through FCM technique and fluorescent microscope (145±59 / 1×105 vs 298±72 / 1×105; 755±158/ 5×105 vs 1598±227/ 5×105; n=9, p<0.05). FCM showed hUC-MSC could inhibit CD69 expression on PHA and PMA activated T cells from 22. 6±5.2% to 7.8±3.5% (p<0.01), further more such inhibitation affected both CD4+ and CD8+ T-cell subsets. A dose dependence of inhibitory effect of hUC-MSC on T-lymphocyte proliferation induced by PHA was also found by MTT. The NOD/SCID mouse model of cotransplantation showed the survival rate of NOD/SCID mouse after cotransplantation was improved from 44.4% to 88.9% as compared with MNCs transplantation alone, and the extent of engraftment of umbilical cord blood cells (chimerism of human CD45+ cells) in the bone marrow of NOD/SCID mouse were also increased from 8.97±1.85% to 35.72±15.41% measured by FCM.Conclusion1. A simple method to isolate and culture-expand hUC-MSCs from human umbilical cord was established successfully. The number of hearvested hUC-MSCs was appropriate for therapeutic purpose.2. hUC-MSC had the basic biological and phenotypic characteristics of MSC, and its capacity of high proliferation was quite outstanding.3. Three methods to preserve hUC-MSCs were primally explored which might do favor to establishing bank of hUC-MSC in the future.4. hUC-MSC had the capacity of hematpoietic-support in a long term through maintaining suvival and preventing differentiation of hematpoietic stem/progenitor cells.5. hUC-MSC could augment the homing and engraftment of hematpoietic stem/progenitor cells which would accelerate the reconstrution of hematopoiesis.6. hUC-MSC had a dose dependent inhibitory effect on T lymphocyte activation and proliferation, and such effect was not selective. These results indicated the immunomodulatory capacity of hUC-MSC which should open the horizons for hUC-MSCs used in much more allogeneic hemotopoietic stem cell transplantations to prevent and treat engraftment rejection and GVHD.7. The NOD/SCID mouse model of cotransplantation confirmed the capacity of hUC-MSC to augment the engraftment and reconstrution of hematopoiesis following a significant improvement of transplantation outcome.
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