Protective Effect of Thrombopoietin on Myocardial Cell in Vitro

Objectives To investigate the protective effect of thrombopoietin (TPO) on myocardial cells in vitro. Methods H9C2 cell line was maintained in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% calf serum. Beating cells from heart ventricles of neonatal heart were cultured at an in vitro system. Apoptosis of the cell line above was induced by treatment of doxorubicin (DOX) and was blocked by TPO. Cell survival rate of H9C2 cell was measured by the MTT assay. Changes of beating rate of neonatal myocardial cells were captured by digital camera and beating rate was calculated. Flow cytometry was employed to study anti-apoptotic effect of TPO by staining JC-1 protein to H9C2 cell. Results MTT assay demonstrated that doxorubicin reduced cell survival rate by 73.8%±1.1%, 50 ng·mL-1 and 100 ng·mL-1 TPO increased cell survival rate by 84.6%±3.6% (P<0.05), 86%±4% (P<0.01) at a dose-dependent manner. Beating rate of primary neonatal myocardial cells also decreased to 15%±8% at 48 h, 100 ng·mL-1 TPO improved beating rate to 48%±11% (P<0.01). TPO decreased apoptotic rate from 19%±9% to 11%±6% (P<0.05). Conclusions TPO has protective effect on myocardial cells in vitro. Anti-apoptosis is one of the mechanisms by which TPO protects injured heart.     

Effect of hepatoma H22 on lymphatic endothelium in vitro

AIM: To determine the effect of metastatic hepatoma cells on lymphangioma-derived endothelium, and to establish in vitro model systems for assessing metastasis-related response of lymphatic endothelium.METHODS: Benign lymphangioma, induced by intraperitoneal injection of the incomplete Freund‘s adjuvant in BALB/c mice, was embedded in fibrin gel or digested and then cultured in the conditioned medium derived from hepatoma H22. Ught and electron microscopy, and the b-answell migration assay were used to determine the effect of H22 on tissue or cell culture. Expressions of Fit-4, c-Fos, proliferating cell nuclear antigen (PCNA), and inducible nitric oxide synthase (iNOS) in cultured cells, and content of nitric oxide in culture medium were also examined.RESULTS: The embedded lymphangioma pieces gave rise to array of capillaries, while separated cells from lymphangioma grew to a cobblestone-like monolayer. H22 activated growth and migration of the capillaries and cells, induced expressions of Flt-4, c-Fos, PCNA and iNOS in cultured cells, and significantly increased the content of NO in the culture medium.CONCLUSION: Lymphangioma-derived cells keep the differentiated phenotypes of lymphatic endothelium, and the models established in this study are feasible for in vitro study of metastasis-related response of lymphatic endothelium.     

Evaluation of diffusion in gel entrapment cell culture within hollow fibers

AIM: To investigate diffusion in mammalian cell culture by gel entrapment within hollow fibers. METHODS: Freshly isolated rat hepatocytes or human oral epidermoid carcinoma (KB) cells were entrapped in type I collagen solutions and statically cultured inside microporous and ultrafiltration hollow fibers. During the culture time collagen gel contraction, cell viability and specific function were assessed. Effective diffusion coefficients of glucose in cell-matrix gels were determined by lag time analysis in a diffusion cell. RESULTS: Significant gel contractions occurred in the collagen gels by entrapment of either viable hepatocytes or KB cells. And the gel contraction caused a significant reduction on effective diffusion coefficient of glucose. The cell viability assay of both hepatocytes and KB cells statically cultured in hollow fibers by collagen entrapment further confirmed the existence of the inhibited mass transfer by diffusion. Urea was secreted about 50% more by hepatocytes entrapped in hollow fibers with pore size of 0.1 μm than that in hollow fibers with MWCO of 100 ku. CONCLUSION: Cell-matrix gel and membrane pore size are the two factors relevant to the limited mass transfer by diffusion in such gel entrapment of mammalian cell culture.     

Liver-specific gene expression in mesenchymal stem cells is induced by liver cells

AIM: The origin of putative liver cells from distinct bone marrow stem cells, e.g. hematopoietic stem cells or multipotent adult progenitor cells was found in recent in vitro studies. Cell culture experiments revealed a key role of growth factors for the induction of liver-specific genes in stem cell cultures. We investigated the potential of rat mesenchymal stem cells (MSC) from bone marrow to differentiate into hepatocytic cells in vitro. Furthermore, we assessed the influence of cocultured liver cells on induction of liver-specific gene expression. METHODS: Mesenchymal stem cells were marked with green fluorescent protein (GFP) by retroviral gene transduction. Clonal marked MSC were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with SCF, HGF, EGF, and FGF-4 alone, or in presence of freshly isolated rat liver cells. Cells in cocultures were harvested and GFP+ or GFP-cells were separated using fluorescence activated cell sorting. RT-PCR analysis for the stem cell marker Thy1 and the hepatocytic markers CK-18, albumin, CK-19, and AFP was performed in the different cell populations. RESULTS: Under the specified culture conditions, rat MSC cocultured with liver cells expressed albumin-, CK-18, CK-19, and AFP-RNA over 3 weeks, whereas MSC cultured alone did not show liver specific gene expression. CONCLUSION: The results indicate that (1) rat MSC from bone marrow can differentiate towards hepatocytic lineage in vitro, and (2) that the microenvironment plays a decisive role for the induction of hepatic differentiation of rMSC.     

Interaction between enteric epithelial cells and Peyer＇s patch lymphocytes in response to Shigella lipopolysaccharide： Effect on nitric oxide and IL-6 release

AIM: To investigate the effect of interaction between enteric epithelial cells and lymphocytes of Peyer's patch on the release of nitric oxide (NO) and IL-6 in response to Shigella lipopolysaccharide (LPS). METHODS: Human colonic epithelial cells (Caco-2) were mixed cocultured with lymphocytes of Peyer's patch from wild-type (C57 mice) and inducible NO synthase knockout mice, and challenged with Shigella F2a-12 LPS. Release of NO and mIL-6 was measured by Griess colorimetric assay and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: In the absence of LPS challenge, NO was detected in the culture medium of Caco-2 epithelial cells but not in lymphocytes of Peyer's patch, and the NO release was further up-regulated in both cocultures with lymphocytes from either the wild-type or iNOS knockout mice, with a significantly higher level observed in the coculture with iNOS knockout lymphocytes. After Shigella F2a-12 LPS challenge for 24-h, NO production was significantly increased in both Caco-2 alone and the coculture with lymphocytes of Peyer's patch from the wild-type mice but not from iNOS knockout mice. LPS was found to stimulate the release of mIL-6 from lymphocytes, which was suppressed by coculture with Caco-2 epithelial cells. The LPS-induced mIL-6 production in lymphocytes from iNOS knockout mice was significantly greater than that from the wild-type mice. CONCLUSION: Lymphocytes of Peyer's patch maintain a constitutive basal level of NO production from the enteric epithelial cell Caco-2. LPS-induced mIL-6 release from lymphocytes of Peyer's patch is suppressed by the cocultured epithelial cells. While no changes are detectable in NO production in lymphocytes from both wild-type and iNOS knockout mice before and after LPS challenge, NO from lymphocytes appears to play an inhibitory role in epithelial NO release and their own mIL-6 release in response to LPS.     

Inhibitory effects of dobutamine on human gastric adenocarcinoma

AIM:To explore the inhibitory effects of dobutamine on gastric adenocarcinoma cells.METHODS:Dobutamine was used to treat gastric adenocarcinoma cells(SGC-7901)and cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay.The effects of dobutamine combined with cisplatin on cell viability were also analyzed.Cell migration was studied using the wound healing assay,and cell proliferation was analyzed using the colony formation assay.A cell invasion assay was carried out using Transwell cell culture chambers.The cell cycle and cell apoptosis were analyzed by flow cytometry.Western blot and immunocytochemistry were performed to determine the expression of Yes-associated protein(YAP)in treated cells.RESULTS:Dobutamine significantly inhibited cell growth,migration,cell colony formation,and cell invasion into Matrigel.Dobutamine also arrested the cell cycle at G1/S phase,and increased the rate of apoptosis of gastric adenocarcinoma cells.The expression ofYAP was detected mainly in the nucleus in the absence of dobutamine.However,reduced expression of phosphorylated YAP was mainly found in the cytosol following treatment with dobutamine.CONCLUSION:Dobutamine has significant inhibitory effects on gastric adenocarcinoma cells and may be used in neoadjuvant therapy not only for gastric cancer,but also for other tumors.     

HepG2 cells support viral replication and gene expression of hepatitis C virus genotype 4 in vitro

AIM: To establish a cell culture system with long-term replication of hepatitis C virus (HCV) genome and expression of viral antigens in vitro. METHODS: HepG2 cell line was tested for its susceptibility to HCV by incubation with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various time points during the culture. Culture supernatant was tested for its ability to infect naive cells. The presence of minus (antisense) RNA strand, and the detection of core and El antigens in cells were examined by RT-PCR and immunological techniques (flow cytom-etry and Western blot) respectively. RESULTS: The intracellular HCV RNA was first detected on d 3 after infection and then could be consistently detected in both cells and supernatant over a period of at least three months. The fresh cells could be infected with supernatant from cultured infected cells. Flow cytometric analysis showed surface and intracellular HCV antigen expression using in house made polyclonal antibodies (anti-core, and anti-El). Western blot analysis showed the expression of a cluster of immunogenic peptides at molecular weights extended between 31 and 45 kDa in an one month old culture of infected cells whereas this cluster was undetectable in uninfected HepG2 cells. CONCLUSION: HepG2 cell line is not only susceptible to HCV infection but also supports its replication in vitro. Expression of HCV structural proteins can be detected in infected HepG2 cells. These cells are also capable of shedding viral particles into culture media which in turn become infectious to uninfected cells.     

Effects of dendritic cells from cord blood CD34^＋ cells on human hepatocarcinoma cell line BEL-7402 in vitro and in SCID mice

AIM: To develop a cancer vaccine of dendritic cells derived from human cord blood CD34+ cells and to investigate its cytotoxicity on human hepatocarcinoma cells in vitro and in sever combined immunodeficiency (SCID) mice. METHODS: Lymphocytes from cord blood or peripheral blood were primed by DCs, which were derived from cord blood and pulsed with whole tumor cell lysates. Nonradiative neutral red uptake assay was adopted to detect the cytotoxicity of primed lymphocytes on human hepatocartinoma cell line BEL-7402 in vitro. The anti-tumor effect of primed lymphocytes in vivo was detected in SCID mice, including therapeutic effect and vaccination effect. RESULTS: The cytotoxicity of DC vaccine primed lymphocytes from cord blood or peripheral blood on human hepatocarcinoma cell line BEL-7402 was significantly higher than that of unprimed lymphocytes in vitro (44.09% vs 14.69%, 47.92% vs 19.44%, P<0.01). There was no significant difference between the cytotoxicity of primed lymphocytes from cord blood and peripheral blood (P>0.05). The tumor growth rate and tumor size were smaller in SCID mice treated or vaccinated with primed lymphocytes than those with unprimed lymphocytes. SCID mice vaccinated with primed lymphocytes had a lower tumor incidence (80% vs 100%, P<0.05) and delayed tumor latent period compared with mice vaccinated with unprimed lymphocytes (11d vs 7 d,P<0.01). CONCLUSION: Vaccine of cord blood derived-DCs has an inhibitory activity on growth of human hepatocarcinoma cells in vitro and in SCID mice. The results also implicate the potential role of cord blood derived-DC vaccine in clinical tumor immunotherapy.     

A rapid and efficient method to express target genes in mammalian cells by baculovirus

AIM: To investigate the modification of baculovirus vectorand the feasibility of delivering exogenous genes intomammalian cells with the culture supernatant of Spodopterafrugiperta (Sf9) cells infected by recombinant baculoviruses.METHODS: Two recombinant baculoviruses (BacV-CMV-EGFPA,BacV-CMV-EGFPB) containing CMV-EGFP expressioncassette were constructed.HepG2 cells were directlyincubated with the culture supernatant of Sf9 cells infectedby recombinant baculoviruses,and reporter gene transferand expression efficiencies were analyzed by flow cytometry(FCM).The optimal transduction conditions were investigatedby FCM assay in HepG2 cells.Gene-transfer and expressionefficiencies in HepG2 or CVl cells by baculovirus vectorswere compared with lipofectAMINE,recombinant retrovirusand vaccinia virus expression systems.Twenty differentmammalian cell lines were used to investigate the feasibilityof delivering exogenous genes into different mammalian cellswith the culture supernatant of infected Sf9 cells.RESULTS: CMV promoter could directly express reportergenes in Sf9 cells with a relatively low efficiency.Targetcells incubated with the 1:1 diluted culture supernatant(moi=50) for 12 h at 37℃ could achieve the highesttransduction and expression efficiencies with leastimpairment to cell viability.Under similar conditions thebaculovirus vector could achieve the highest gene-transferand expression efficiency than lipofectAMINE,recombinantretrovirus and vaccinia virus expression systems.Mostmammalian cell lines could be transduced with recombinantbaculovirus.In primate adherent culture cells therecombinant baculovirus could arrive the highest infectionand expression efficiencies,but it was not very satisfactoryin the cell lines from mice and suspended culture cells.CONCLUSION: Mammalian cells incubated with the culturesupernatant of infected Sf9 cells could serve as a veryconvenient way for rapid and efficient expression of foreigngenes in mammalian cells,but it might be more suitable forprimate adherent culture cells.     

Fas ligand expression in colon cancer： A possible mechanism of tumor immune privilege

AIM: To detect the expression of Fas ligand (FasL) in colon cancer tissues and cell lines and analyze the function of FasL-expressing colon cancer cells in inducing Fas-sensitive T lymphocyte apoptosis. METHODS: Ninety surgically resected colon cancer tissues and 15 hepatic metastasis specimens were investigated by immunohistochemical method with normal colon mucosa and colon adenoma as control. The relationship between FasL expression and pathologic features was also analyzed. FasL expression of 4 colon cancer cell lines, SW620, Lovo, LS-174T and SW1116, were detected by Western blotting assay. The function of FasL expressed on colon cancer cells was determined by coculture assay with Jurkat T lymphocytes, the apoptotic rate of which was detected by flow cytometry assay. RESULTS: Fifty-six (62.22%) cases of all the 90 colon cancer tissues and all (100%) the liver metastasis specimens expressed FasL, significantly higher than normal colon mucosa and colonic adenoma. Higher expression of FasL was found in more advanced stage of colon cancer and in cancer tissues with lymphatic or hepatic metastasis. All the colon cancer cell lines were found to express FasL. After coculture with the SW1116 cells for 24 h with an effector: target ratio 10:1, the rate of apoptosis of Jurkat cells rose from 1.9% to 21.0%. CONCLUSION: The expression of FasL is upregulated in colon cancer and the functionally expressed FasL can induce apoptosis of Fas-expressing T lymphocytes.     

Rapid ex vivo expansion of human umbilical cord hematopoietic progenitors using a novel culture system.

Cell numbers limit the widespread clinical use of cord blood (CB) for gene therapy and marrow replacement in adults; a simple and effective method for ex vivo expansion of CB primitive progenitor cells (PPC) is required. Recently, the combination of thrombopoietin (TPO) and Flk-2/Flt-3 ligand (FL-2) was reported to support slow proliferation of CB-PPC in stroma-free liquid culture. We established a novel culture system in which the murine stromal cell line HESS-5 dramatically supports the rapid expansion of cryopreserved CB-PPC in synergy with TPO/FL-2. Furthermore, while HESS-5 cells directly adhered to human progenitors during culture, the cultured human cells could easily be harvested without contamination by HESS-5 cells. Within 7 days of culture, a 100-fold increase in CD34bright/CD38dim cells was obtained in serum-containing culture. When HESS-5 cells were physically separated from human progenitor cells in the presence of TPO/FL-2, synergy was blocked, suggesting that HESS-5 cells support proliferation of PPC by direct cell-to-cell interaction. The hematopoietic-supportive effects of this xenogeneic coculture system were then assessed in a very short-term (5 days) serum-free culture. Expansion was further enhanced by addition of stem cell factor (SCF) or interleukin-3 (IL-3). As a result, a 50- to 100-fold increase in CD34bright/CD38dim cells was noted. Colony-forming units in culture (CFU-C) and mixed colonies (CFU-GEMM) were enhanced by 10- to 30-fold and 10- to 20-fold, respectively. Moreover, generation of long-term-culture-initiating cells (LTC-IC) from CD34bright/CD38dim cells was amplified by 25-fold. The severe-combined immunodeficient (SCID) mouse-repopulating cell (SRC) assay confirmed extensive ability of the expanded cells to reconstitute long-term hematopoiesis. These results indicate that this xenogeneic coculture system, in combination with human cytokines, can rapidly generate PPC from cryopreserved CB.     

Stromal cell-dependent ex vivo expansion of human cord blood progenitors and augmentation of transplantable stem cell activity

In vitro maintenance and expansion of human hematopoietic stem cells is crucial for many clinical applications. Thrombopoietin (TPO) and flt3/flk2 ligand (FL) have been suggested to support the proliferation of primitive hematopoietic progenitors and the expansion of transplantable stem cells in culture. In this study, we examined the synergistic effects of the murine stromal cell line MS-5 and a combination of the two cytokines, TPO and FL, on the ex vivo expansion of human cord blood primitive progenitors and transplantable stem cells. A monolayer of MS-5 cells with TPO/FL synergistically supported a more than 600-fold expansion of human cord blood CD34+ cells and CD34+CD38- cells in 2 weeks of culture. Colony-forming unit in culture (CFU-C) and 5-week and 8-week cobblestone area-forming cells (CAFC) were also expanded approximately 300-, 4- and 13-fold, respectively. When MS-5 cells were physically separated from progenitors by a Transwell filter, the synergy was reduced to a quarter of the control, suggesting that direct cell-cell contact between MS-5 cells and progenitors is required for maximum expansion. The severe-combined immunodeficient (scid) mouse-reconstituting cell (SRC) assay demonstrated the slight augmentation of transplantable stem cell activity in culture. These results indicated that MS-5 cells provide a milieu that stimulates the proliferation of primitive progenitors including transplantable stem cells.     

Serum-free coculture system for ex vivo expansion of human cord blood primitive progenitors and SCID mouse-reconstituting cells using human bone marrow primary stromal cells

OBJECTIVE: In an attempt to maintain and expand human stem cells, many investigators have used xenogeneic, especially murine, stromal cells and fetal calf serum. Because of the possible transmission of infectious diseases, however, the safety of the delivery of grafts expanded in culture using xenogeneic cells and serum has been debated. Using primary human marrow stromal cells, we established a novel serum-free culture system to expand human primitive progenitors and transplantable stem cells. MATERIALS AND METHODS: Cord blood CD34(+) cells were cultured on a monolayer of human primary marrow stromal cells in the presence of thrombopoietin (TPO), flt3/flk2 ligand (FL), and/or stem cell factor (SCF) under serum-free conditions. After 2 or 4 weeks of culture, cells were examined for clonogenic progenitors and severe combined immunodeficient disorder (SCID) mouse-reconstituting cells (SRC). RESULTS: In the presence of TPO, FL, and SCF, marrow stromal cells supported more than a 100- and 1,000-fold expansion of CD34(+) cells and colony-forming units in culture after 2 and 4 weeks of incubation, respectively. In addition, cobblestone area-forming cells were expanded more than 18- and 60-fold after 2 and 4 weeks of culture, respectively. Furthermore, SRC assay demonstrated augmented engraftment by cultured cells. CONCLUSION: This ex vivo expansion system should prove valuable in clinical settings in which stromal cells are available from recipients or stem cell donors.     

All-trans retinoic acid (ATRA) enhances maintenance of primitive human hematopoietic progenitors and skews them towards myeloid differentiation in a stroma-noncontact culture system

OBJECTIVE: We have previously shown that hematopoietic progenitor cells (HPCs) from umbilical cord blood (UCB) can be maintained in a cytokine-supplemented stroma-noncontact (SNC) system. Here, we tested if all-trans retinoic acid (ATRA), known to improve expansion of murine hematopoietic stem cells, would enhance human HPC maintenance in a SNC culture system. METHODS: CD34+CD38-Lin- cells from UCB were cultured in transwells above AFT024 in the presence of Flt-3 ligand (FLT) and thrombopoietin (TPO), with or without ATRA. Total nucleated cells (TNC), colony-forming units (CFUs), long-term culture-initiating cells (LTC-ICs), myeloid-lymphoid initiating cells (ML-ICs) and SCID repopulating cells (SRCs) were evaluated 1 to 5 weeks after culture. RESULTS: All-trans retinoic acid (1 mumol/L) reduced expansion of CD34+CD38-Lin- TNC and CFUs after 2 to 5 weeks of culture. However, it significantly increased LTC-IC expansion after 1 to 3 and, even more so, 5 weeks of culture. ATRA also increased recovery of more primitive ML-ICs and SRCs. Increased HPC recovery appeared dependent on the presence of stromal cells, as LTC-IC expansion was significantly reduced when ATRA was added to stroma-free cultures. CONCLUSION: All-trans retinoic acid increases expansion of early HPCs in a stromal cell-dependent fashion.     

Human placenta-derived mesenchymal progenitor cells support culture expansion of long-term culture-initiating cells from cord blood CD34+ cells

OBJECTIVE: Allogeneic transplantation with umbilical cord blood (UCB) in adult recipients is limited mainly by a low CD34+ cell dose. To overcome this shortcoming, human placenta as a novel source of human mesenchymal progenitor cell (MPC) was incorporated in an attempt to expand CD34+ cells from UCB in vitro. MATERIALS AND METHODS: Human placenta MPC was isolated and characterized by morphologic, immunophenotypical, and functional analysis. UCB CD34+ cells were expanded by coculture with placental MPC. Suitable aliquots of cells were used to monitor cell production, clonogenic activity, and long-term culture-initiating culture (LTC-IC) output. Finally, the immunoregulatory effect of placental MPC was evaluated by T-cell proliferation assay. RESULTS: In its undifferentiated state, placental MPC displayed fibroblastoid morphology; was CD73, CD105, CD29, CD44, HLA-ABC, and CD166 positive; produced fibronectin, laminin, and vimentin; but was negative for CD14, CD31, CD34, CD45, HLA-DR, and alpha-smooth muscle actin. Functionally, it could be induced into adipocytes, osteocytes, and chondrocytes. In vitro expansion of UCB hematopoietic cells, when cocultured with placental MPC in the presence of cytokines, was significantly enhanced: CD34+ cells by 14.89 +/- 2.32 fold; colony-forming cell (CFC) by 36.73 +/- 5.79 fold; and LTC-IC by 7.43 +/- 2.66 fold. Moreover, placental MPC could suppress T-cell proliferation induced by cellular stimuli. CONCLUSION: These results strongly suggest that human placental MPC may be a suitable feeder layer for expansion of hematopoietic progenitors from UCB in vitro.     

Homing defect of cultured human hematopoietic cells in the NOD/SCID mouse is mediated by Fas/CD95

OBJECTIVE: To determine the bone marrow homing efficiency (20 hours) of cultured compared to noncultured umbilical cord blood (UCB)-derived human hematopoietic cells in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse, and to explain the difference in homing between these populations. METHODS: Human UCB CD34+ cells were cultured for up to 5 days, reselected, and used for transplantation, phenotype analysis, and functional studies, including adhesion and trans-endothelial migration assays. Seeding of CD34+ cells was measured after labeling of cells with 111-Indium, while homing of colony-forming cells (CFC) and SCID-repopulating (SRC) cells was determined using functional assays. RESULTS: Short-term culture was associated with a decrease in the 20-hour homing of CD34+ cells, CFC, and SRC to the BM. Although cultured compared to noncultured cells showed increased expression and function (adhesion/migration) of several cell adhesion molecules described to play a role in homing and engraftment, culture also induced expression of Fas/CD95 and rendered cells more susceptible to apoptosis. Finally, we demonstrate that the level of Fas/CD95 on cultured cells was inversely related to the ability of CFC to home to the BM, and that the homing of cultured CFC could be restored by incubating cells prior to transplantation with Fas/CD95-blocking mAb ZB4. CONCLUSION: These data implicate Fas/CD95 in the homing defect of cultured human hematopoietic cells in the NOD/SCID transplant model and suggest that prevention of apoptosis may be an important strategy to improve engraftment of ex vivo-manipulated HSC in a clinical setting.     

Platelet-derived growth factor promotes ex vivo expansion of CD34+ cells from human cord blood and enhances long-term culture-initiating cells,non-obese diabetic/severe combined immunodeficient repopulating cells and formation of adherent cells

Platelet-derived growth factor (PDGF) is a major mitogen for connective tissue cells. In this study, we investigated the effects and mechanism of PDGF on the ex vivo expansion of cord blood CD34+ cells. Our data demonstrated that among various cytokine combinations of thrombopoietin (TPO), interleukin 1 beta (IL-1beta), IL-3, IL-6 and Flt-3 ligand (Flt-3L), TPO + IL-6 + Flt-3L was most efficient in promoting the expansion of CD34+ cells, CD34+CD38- cells, mixed-lineage colony-forming units (CFU-GEMM) and long-term culture-initiating cells (LTC-IC) by 21.7 +/- 5.00-, 103 +/- 27.9-, 10.7 +/- 7.94- and 6.52 +/- 1.51-fold, respectively, after 12-14 d of culture. The addition of PDGF increased the yield of these early progenitors by 45.0%, 66.5%, 45.1% and 79.8% respectively. More significantly, PDGF enhanced the engraftment of human CD45+ cells and their myeloid subsets (CD33+, CD14+ cells) in non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) mice. The expression of PDGF receptor (PDGFR)-beta was not detectable in fresh CD34+ cells but was upregulated after culture for 3 d. PDGF also enhanced the development of adherent cells/clusters that expressed the endothelial markers VE-cadherin and CD31. These findings suggest that PDGF is an effective cytokine for the ex vivo expansion of early stem and progenitor cells. The mechanism could be mediated by PDGFR-beta on committed CD34+ progenitor cells and/or secondary to the stimulation of autologous, stromal feeder cells.     

Optimization of multidrug resistance 1 gene transfer confers chemoprotection to human hematopoietic cells engrafted in immunodeficient mice

OBJECTIVE: To investigate whether an optimization of MDR1 gene transfer protocol would result in stable hematopoietic stem cell (HSC) engraftment and myeloprotection in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice after paclitaxel chemotherapy. METHODS: We transplanted freshly isolated CD34+ cells or MDR1-transduced CD34+ cells derived from human umbilical cord blood (UCB) into sublethally irradiated NOD/SCID mice. Twenty-eight days after transplantation, mice received paclitaxel chemotherapy and peripheral blood (PB) was collected for analysis of WBC, RBC and PLT counts once every week. RESULTS: We found that MDR1-transduced human hematopoietic cells could facilitate hematopoietic recovery and completely reconstitute hematopoiesis in mice as well as freshly isolated CD34+ cells. Mice transplanted with MDR1-transduced human hematopoietic cells were protected from paclitaxel chemotherapy with higher survival rate and higher level of WBC counts and RBC counts compared with mice transplanted with untransduced HSCs. We also demonstrated that hematopoietic cells transduced with MDR1 gene were enriched in vivo after paclitaxel chemotherapy determined by the higher percentage of human Rh-123(dull) CD45+ cells in bone marrow of mice. CONCLUSION: Our results demonstrated successful chemoprotection against myelosuppression in mice by MDR1-transduced repopulating human hematopoietic cells with an optimized transduction protocol.     

Ex vivo expansion of human adult stem cells capable of primary and secondary hemopoietic reconstitution

OBJECTIVE: Ex vivo expansion of human hemopoietic stem cells (HSC) is an important issue in transplantation and gene therapy. Encouraging results have been obtained with cord blood, where extensive amplification of primitive progenitors was observed. So far, this goal has been elusive with adult cells, in which amplification of committed and mature cells, but not of long-term repopulating cells, has been described. METHODS: Adult normal bone marrow (BM) and mobilized peripheral blood (MPB) CD34(+) cells were cultured in a stroma-free liquid culture in the presence of Flt-3 ligand (FL), thrombopoietin (TPO), stem cell factor (SCF), interleukin-6 (IL-6), or interleukin-3 (IL-3). Suitable aliquots of cells were used to monitor cell production, clonogenic activity, LTC-IC output, and in vivo repopulating capacity. RESULTS: Here we report that BM and MPB HSC can be cultured in the presence of FL, TPO, SCF, and IL-6 for up to 10 weeks, during which time they proliferate and produce large numbers of committed progenitors (up to 3000-fold). Primitive NOD/SCID mouse repopulating stem cells (SRC) are expanded sixfold after 3 weeks (by limiting dilution studies) and retain the ability to repopulate secondary NOD/SCID mice after serial transplants. Substitution of IL-6 with IL-3 leads to a similarly high production of committed and differentiated cells but only to a transient (1 week) expansion of SRC(s), which do not possess secondary repopulation capacity. CONCLUSION: We report evidence to show that under appropriate culture conditions, adult human SRC can also be induced to expand with limited differentiation.     

Development of functional human blood and immune systems in NOD/SCID/IL2 receptor {gamma} chain(null) mice

Here we report that a new nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse line harboring a complete null mutation of the common cytokine receptor gamma chain (NOD/SCID/interleukin 2 receptor [IL2r] gamma(null)) efficiently supports development of functional human hemato-lymphopoiesis. Purified human (h) CD34(+) or hCD34(+)hCD38(-) cord blood (CB) cells were transplanted into NOD/SCID/IL2rgamma(null) newborns via a facial vein. In all recipients injected with 10(5) hCD34(+) or 2 x 10(4) hCD34(+)hCD38(-) CB cells, human hematopoietic cells were reconstituted at approximately 70% of chimerisms. A high percentage of the human hematopoietic cell chimerism persisted for more than 24 weeks after transplantation, and hCD34(+) bone marrow grafts of primary recipients could reconstitute hematopoiesis in secondary NOD/SCID/IL2rgamma(null) recipients, suggesting that this system can support self-renewal of human hematopoietic stem cells. hCD34(+)hCD38(-) CB cells differentiated into mature blood cells, including myelomonocytes, dendritic cells, erythrocytes, platelets, and lymphocytes. Differentiation into each lineage occurred via developmental intermediates such as common lymphoid progenitors and common myeloid progenitors, recapitulating the steady-state human hematopoiesis. B cells underwent normal class switching, and produced antigen-specific immunoglobulins (Igs). T cells displayed the human leukocyte antigen (HLA)-dependent cytotoxic function. Furthermore, human IgA-secreting B cells were found in the intestinal mucosa, suggesting reconstitution of human mucosal immunity. Thus, the NOD/SCID/IL2rgamma(null) newborn system might be an important experimental model to study the human hemato-lymphoid system.