Long-term ex vivo expansion of human fetal liver primitive haematopoietic progenitor cells in stroma-free cultures

Successful expansion of haematopoietic cells in ex vivo cultures will have important applications in transplantation, gene therapy, immunotherapy and potentially also in the production of non-haematopoietic cell types. Haematopoietic stem cells (HSC), with their capacity to both self-renew and differentiate into all blood lineages, represent the ideal target for expansion protocols. However, human HSC are rare, poorly characterized phenotypically and genotypically, and difficult to test functionally. Defining optimal culture parameters for ex vivo expansion has been a major challenge. We devised a simple and reproducible stroma-free liquid culture system enabling long-term expansion of putative haematopoietic progenitors contained within frozen human fetal liver (FL) crude cell suspensions. Starting from a small number of total nucleated cells, a massive haematopoietic cell expansion, reaching > 1013-fold the input cell number after approximately 300 d of culture, was consistently achieved. Cells with a primitive phenotype were present throughout the culture and also underwent a continuous expansion. Moreover, the capacity for multilineage lymphomyeloid differentiation, as well as the recloning capacity of primitive myeloid progenitors, was maintained in culture. With its better proliferative potential as compared with adult sources, FL represents a promising alternative source of HSC and the culture system described here should be useful for clinical applications.     

Ex vivo expansion of megakaryocyte progenitor cells from normal bone marrow and peripheral blood and from patients with haematological malignancies

A number of haematological and non-haematological malignancies can be successfully treated using high-dose chemotherapy +/- irradiation followed by haematopoietic progenitor cell transplantation. Post transplant, thrombocytopenia and neutropenia always occur and patients require platelet transfusions. It may be possible to reduce the period of thrombocytopenia by re-infusion of ex vivo expanded megakaryocyte progenitors (MP), derived from the progenitor cell graft. We have investigated the expansion of MP from CD34+ enriched cells from normal bone marrow (NBM) and peripheral blood (PB) and remission BM or PB samples from patients with haematological malignancies. CD34+ cells were cultured in serum-free medium supplemented with thrombopoietin (TPO), interleukin 1 (IL-1), IL-6 and stem cell factor (SCF) for 7 d, then cell proliferation was assessed by flow cytometry using lineage-specific markers. It was possible to significantly expand the number of MP cells from all sources. There were no major differences in yields of MP from normal BM or PB, or BM from multiple myeloma and non-Hodgkin's lymphoma patients. However, expansion of MP in acute myeloid leukaemia samples was lower than all other samples and the number of megakaryocyte colony-forming units was reduced. Several cytokine combinations were evaluated to optimize MP expansion from NBM. Equivalent yields of MP were obtained using TPO and one of IL-1, IL-3, granulocyte-macrophage colony-stimulating factor or SCF, suggesting that large cytokine combinations are not necessary for this procedure. It should be possible to scale up the culture conditions described to produce effective MP doses for clinical transplantation.     

Transforming growth factor-beta 1 delays formation of granulocyte-macrophage colony-forming cells, but spares more primitive progenitors during ex vivo expansion of CD34+ haemopoietic progenitor cells

Ex vivo culture and expansion of autologous haemopoietic transplants has been developed to improve tumour cell purging and accelerate haemopoietic reconstitution by transplantation of increased progenitor cell numbers. We studied the effect of the negative haemopoietic regulator, transforming growth factor beta-1 (TGF-beta 1) on primitive precursors during ex vivo expansion of CD34⁺ cells. When added directly to methylcellulose colony-forming assays, TGF-beta 1 potently suppressed the development of granulocyte-macrophage colonies from CD34⁺ enriched peripheral blood progenitor cells (80–90% inhibition). In contrast, expansion of total nucleated cells and granulocyte-macrophage colony-forming cells (GM-CFC) from CD34⁺ progenitors in liquid culture in the presence of stem cell factor (SCF), interleukin (IL)-1 beta, IL-3, IL-6 and erythropoietin (EPO) was inhibited to 32–65% of control culture levels within 14 d when TGF-beta 1 was added, and still produced an average 3.3-fold absolute amplification of GM-CFC. The inhibitory effect of TGF-beta 1 on GM-CFC generation was reversed when it was washed out on day 6 of ex vivo expansion cultures, and total numbers of GM-CFC generated from expansion cultures then reached levels of untreated controls by day 16. Long-term bone marrow culture-initiating cell (LTCIC) numbers were preserved, at least at input levels, over a culture period of 14 d both in control and TGF-beta-1-treated expansion cultures. These findings suggest that TGF-beta 1, a cytokine which induces apoptosis or terminal differentiation in a number of malignant cell types, may be added to ex vivo expansion cultures without loss of primitive cells from autologous haemopoietic transplants.     

Basic fibroblast growth factor-stimulated ex vivo expansion of haematopoietic progenitor cells from human placental and umbilical cord blood

We investigated whether basic fibroblast growth factor (bFGF) is effective in inducing ex vivo expansion of CD34+ haematopoietic progenitor cells derived from human placental and umbilical cord blood. bFGF significantly promoted the clonal growth of various haematopoietic progenitor cells, including granulocyte-macrophage colony-forming units (CFU-GM), mixed colony-forming units (CFU-Mix) and megakaryocyte colony-forming units (CFU-Meg) under semisolid culture conditions, with an optimal bFGF concentration of 30 ng/ml. CD34+ cells were then cultured in serum-free liquid medium containing various combinations of early-acting cytokines, including thrombopoietin (TPO), stem cell factor (SCF), interleukin 3 (IL-3) and flt3-ligand (FL), with or without bFGF, for 6 and 12 d. Without bFGF, TPO + IL-3, TPO + SCF + FL and TPO +SCF + IL-3 + FL dramatically increased the total numbers of erythroid progenitors, CFU-GM and CFU-Mix by d 12 of culture respectively. However, the addition of bFGF did not promote further proliferation of these progenitors, except for the erythroid progenitors, by d 6 when stimulated with all four cytokines. In contrast, total CFU-Meg numbers were approximately doubled when these cultures were supplemented with bFGF, producing 100- to 120-fold increases compared with the baseline control cultures. These results suggest that bFGF is effective in supporting the generation of megakaryocytic progenitor cells during ex vivo expansion.     

Human neonatal blood: stem cell content, kinetics of CD34+ cell decline and ex vivo expansion capacity

Haemopoietic stem cells are present in fetal blood but their levels decline rapidly in the peripheral circulation of the infant after birth. We previously reported a case of stem cell transplant in a beta-thalassaemia boy using a combination of the cord blood (CB) and neonatal blood (NB) of his sister. This transplant resulted in a successful engraftment. To investigate the possibility of using NB to supplement CB for related transplants, we further characterized stem and progenitor cells and lymphocyte subsets in 20 NB samples, comparing the findings with those in 20 CB samples. Our data showed that NB contained substantial levels of CD34+ cells, CD34+CD38- cells, colony-forming units-granulocyte macrophage (CFU-GM), colony forming units-erythroid (CFU-E), burst forming units-erythroid (BFU-E) and long-term culture initiating cells (LTCIC). NB was similar to CB in the levels of T lymphocytes, but the amounts of B lymphocytes and natural killer cells were higher in CB (P = 0.033, P= 0.001, respectively). The kinetics of CD34+ cells in NB was investigated in serial blood samples obtained from 10 full-term infants at 2, 4, 6, 8, 24 and 48h after birth. CD34+ cells decreased rapidly after birth, declining to only 30% of the 2h level at 48h (P<0012). The rate of decline was greatest in the first 4 h of life. NB from four infants was expanded by culturing the blood samples in the presence of thrombopoietin (Tpo), interleukin 1beta (IL1beta), IL-3, IL-6, flt-3 ligand and stem cell factor (SCF) for 7 d. This resulted in the increase of CD34+ cells, CFU-GM and CFU-MK by 271+/-179, 556+/-385 and 113+/-75 fold respectively. Three of the five samples expanded for 7 d contained LTCIC. These findings suggest that NB might be a supplementary or alternative source of stem cells to CB for transplant. The ethics and practicality of this approach deserve further exploration.     

Apoptosis and megakaryocytic differentiation during ex vivo expansion of human cord blood CD34+ cells using thrombopoietin

Thrombopoietin (TPO), the primary regulator of megakaryocytopoiesis, plays important roles in early haematopoiesis. Previously, we have demonstrated that TPO induces a characteristic pattern of apoptosis during ex vivo expansion of cord blood (CB) CD34+ cells. In this study, we have demonstrated that the TPO-induced apoptotic cells belong to the megakaryocytic (MK) lineage and that initially expanding MK progenitors declined along with the appearance of TPO-induced apoptosis. Human CB CD34+ cells were expanded in serum-free conditions with TPO. Multidimensional flow cytometry using simultaneous measurement of apoptosis and immunophenotyping showed that the TPO-induced apoptotic cells appeared in CD61+ fractions. Immunocytochemical analysis of the fluorescent activated cell-sorted fractions showed that the apoptosis-associated CD44low fraction expressed CD61. Clonogenic assay revealed 7.4 +/- 0.50-fold increase of total megakaryocyte colony-forming units (CFU-MKs) during the initial 9 d. Thereafter, the number of CFU-MKs decreased in parallel with the increase of apoptosis. When the MK colonies were subdivided according to size, the proportion of large colonies progressively decreased, while that of medium and small colonies increased. In particular, from d 6 small colonies became predominant. These results suggested that the MK progenitors matured as they expanded during ex vivo expansion with TPO and then proceeded to apoptosis.     

低浓度增植细胞核抗原反义核酸对脐血CD34+ 造血干/祖细胞体外扩增的影响

目的研究增植细胞核抗原反义核酸(PCNA-ASODN)对脐血造血干/祖细胞体外扩增的调节作用.方法采用免疫磁珠分离技术从新鲜脐带血中分离CD34+细胞,使用低浓度的PCNA-ASODN作用于体外扩增的CD34+细胞,应用流式细胞仪技术检测其扩增后各种细胞的数量,细胞内PCNA的表达及细胞周期的变化情况.结果与对照组相比,加入了低浓度PCNA-ASODN的实验组,其PCNA的表达水平明显较低,表达率为(27.2±3.6)%,且G0/G1期细胞占(55.9±4.4)%,扩增的细胞中CD34+细胞比例增高,达(33.4±3.2)%,而且CD34+CD38-细胞数量达到(57.8±9.9)倍.结论低浓度的PCNA-ASODN抑制CD34+细胞在增殖过程中PCNA表达,减缓扩增过程中的分化作用.     

Prolonged ex vivo culture of cord blood CD34(+) cells facilitates myeloid and megakaryocytic engraftment in the non-obese diabetic severe combined immunodeficient mouse model

A clinical goal for ex vivo expansion of cord blood (CB) CD34(+) cells is to shorten the period of neutropenia and thrombocytopenia following myeloablative therapy and transplantation. Prolongation of cytokine expansion leads to the production of greater numbers of cells, and should have an impact on neutrophil and platelet recovery. Furthermore, expansion of CD34(+) cells should support the continued production of neutrophils and platelets in the 6-week period following transplantation. We tested these hypotheses by characterization of the kinetics (human CD45(+) cells in the blood) and phenotype (CD45, CD34, CD61, CD33, CD19 and CD3) of human engraftment in the non-obese diabetic severe combined immunodeficient mouse (NOD-SCID) following 7 or 14 d of ex vivo expansion of CB CD34(+) cells. Mice transplanted with 14 d cells showed greater percentages of human CD45(+) cells in the blood, bone marrow and spleen than mice transplanted with unexpanded cells or 7 d cells. Prolonging cytokine exposure of CD34(+) cells and transplantation with increasing numbers of input cells facilitated the production of absolute numbers of CD34(+), CD33(+), CD61(+) and CD19(+) cells in vivo. Furthermore, analysis of SCID engrafting potential showed that prolongation of culture duration facilitates in vivo expansion of CD45(+), CD34(+) and CD19(+) cells after transplantation. It is anticipated that prolonged (2 weeks) ex vivo culture of CB will have a beneficial clinical effect.     

Preclinical ex vivo expansion of peripheral blood CD34+ selected cells from cancer patients mobilized with combination chemotherapy and granulocyte colony-stimulating factor

Background and Objectives  Ex vivo peripheral blood progenitor cell (PBPC) expansion has been proposed as a strategy to increase the number of haematopoietic progenitors available for cell transplantation. We have expanded CD34+ cells from PBPCs obtained from four patients with haematological malignancies and one patient with an Ewing's sarcoma.
Materials and Methods  Cells were expanded in the Dideco 'Pluricell system'. After 12 days in culture, we evaluated cell phenotype, total nucleated cells, CD34+ fold increase, cell apoptosis and colony assay of expanded cells. Cell engraftment has been evaluated by transplanting two groups of irradiated non-obese diabetic/severe combined immunodeficient (NOD-SCID) mice with expanded and non-expanded cell populations.
Results  Total nucleated cells and CD34+ cells increased 59·5 and 4·0 times, respectively. The expanded cells were mainly constituted of myeloid and megakaryocytic cells. A significant increase in the number of colony-forming unit–granulocyte macrophage (CFU-GM) was observed in the CFU assay. Ten mice transplanted with expanded cells showed a best overall survival (80%) compared to 10 mice transplanted with non-expanded cells (20%). Human CD45+ cells were detected by flow cytometry and polymerase chain reaction in bone marrow and spleen of transplanted animals. The relative low engraftment level obtained with the expanded cells suggests a loss of SCID repopulating cells maybe due to cell differentiation during expansion.
Conclusions  We have demonstrated the feasibility of the ex vivo expansion of mobilized PBPCs from cancer patients, evidencing a clonal expansion of CFUs and the ability of the expanded cells to engraft the bone marrow and spleen of immunosuppressed mice. The differentiation of the CD34+ stem cell compartment could be further minimized by ameliorating the expansion conditions.     

Changes in c-Kit expression and effects of SCF during differentiation of human erythroid progenitor cells

Summary We analysed c-Kit expression during erythroid differentiation using immunocytochemical staining and flow cytometric analysis. Burst-forming units-erythroid (BFU-E)-derived cell aggregates were identified in methylcellulose cultures containing human umbilical cord blood CD34⁺ cells and were stained by the indirect immunoalkaline phosphatase method. To investigate the changes in levels of cell-surface c-Kit expression, we subjected progenitor cells in liquid culture to flow cytometric analysis. In addition, the effects of stem cell factor (SCF) on cell-surface c-Kit expression were analysed in these two culture systems and the effects of SCF on erythroid colony formation were studied in a methylcellulose culture. c-Kit was expressed on the cell surface from BFU-E to erythroid precursors recognized morphologically as basophilic erythroblasts. Flow cytometric analysis showed that c-Kit expression increased until 6 d in liquid culture, and that decreased expression of c-Kit was associated with the increased expression of glycophorin A. Moreover, SCF increased the size of erythroid colonies when added at days 0, 4 and 8 in methylcellulose cultures. These results indicate that the c-Kit/SCF system still plays in proliferation of erythroid progenitor cells at the colony-forming units-erythroid stage. Finally, expression of c-Kit in erythroid progenitor cells cultured without SCF showed a diffuse pattern on the cell surface, whereas we observed positive c-Kit immuno-reactivity in the region of the Golgi apparatus of these cells cultured with SCF. Flow cytometric analysis also showed that the levels of cell-surface c-Kit expression decreased in the presence of SCF. These results suggest that SCF induced down-modulation of cell-surface c-Kit expression, despite continuous synthesis of c-Kit protein.     

Diaminofluorene stain detects erythroid differentiation in immature haemopoietic cells treated with EPO,IL-3, SCF,TGFbeta1, MIP-1alpha and IFNgamma

We have combined in vitro clonogenic culture and a highly sensitive stain for haemoglobin to compare the influence of EPO, IL-3, SCF, TGFbeta1, MIP-1alpha and IFNgamma, to directly stimulate cells in the progenitor compartment to develop towards the erythroid lineage. Three cell lines were chosen, as they exist developmentally arrested in the progenitor compartment, yet in a pliant state of maturation. HEL (erythroleukaemia) and K562 (CML-derived) cell lines, may, under appropriate stimuli, develop erythroid characters, whilst the third, U937 (as control cell line), may be stimulated by DMSO to differentiate to myeloid cells. After in vitro semi-solid methylcellulose culture with these cytokines, resulting colonies were stained with 2,7-diaminofluorene (DAF), which sensitively stains haemoglobin blue. Haemoglobin production was low in HEL and K562 cells and absent in U937. Cytokine analysis showed varying levels of influence depending on the starting level of cell line maturation. EPO and TGFbeta1 maximally stimulated haemoglobin production in the HEL and K562 cell lines. This differential cytokine stimulation analysis combined with sensitive DAF haemoglobin detection could be applied in the study of many erythropoiesis-deficient patients or primitive erythropoiesis.     

Persistence of residual tumour cells after cytokine-mediated ex vivo expansion of mobilized CD34+ blood cells in multiple myeloma

Mobilized CD34⁺ blood cells were immunomagnetically enriched from leukapheresis products in five multiple myeloma (MM) patients. Thawed samples of selected CD34⁺ cells were cultured for up to 21 d in a liquid and stroma-free culture system with different combinations of recombinant cytokines. The most successful cell expansion was obtained when a combination of rh-IL-1β, rh-IL-3, rh-IL-6, rh-SCF, rh-G-CSF and rh-GM-CSF was used. After 14 d this mixture gave a 120–187-fold overall increase of total nuclear cells and a 4–8-fold overall increase of early CFU-GM numbers. In four patients a very sensitive patient-specific PCR analysis showed the presence of monoclonal cells in the initial leukapheresis products. After immunomagnetic separation a tumour cell depletion of 2–4 logs was observed, although all samples still contained malignant cells. Cell suspensions that were cultured with the most potent cytokine combination showed tumour contamination in two-thirds of evaluable cases at the moment of maximal CFU-GM output. Serial cDNA dilution experiments indicated that the positive PCR results at day 14 reflected the persistence of pre-culture tumour cells rather than in vitro expansion of tumour cells in two cases. This study demonstrates that ex vivo expansion of myeloid precursor cells from mobilized CD34⁺ cells in MM patients does not always result in an effective purging of residual tumour cells. On the other hand, our culture conditions do not seem to favour in vitro expansion of malignant cells, despite the use of a cytokine cocktail that includes potential myeloma growth factors.     

分化抑制培养体系体外扩增脐血造血细胞的研究

目的:探讨分化抑制培养体系对脐血造血细胞体外扩增效应。方法:分化抑制培养体系与脐血单个核细胞(MNC)体外共同培养7 d,检测MNC细胞总数、CFC、CD34 细胞反应扩增效果,并检测CD34 细胞表面归巢相关黏附分子VLA-4(CD49d)、VLA-5(CD49e)、LFA-1(CD11a)、HCAM(CD44)、L-selectin(CD62L)的表达率。结果:分化抑制培养体系组明显扩增脐血MNC细胞总数、CFC、CD34 细胞(均P<0．05),对照组培养脐血MNC细胞总数明显下降,CFC和CD34 细胞完全死亡(均P<0．01)。CD34 细胞表面各黏附分子CD49d、CD44和C1362L表达与扩增前相当(均P>0．05),而CD49e和CD11a表达明显高于扩增前(均P<0．05)。结论:分化抑制培养体系体外显著扩增脐血造血细胞,并且扩增后的造血干(祖)细胞总体上保持其表面归巢相关黏附分子的表达,归巢功能不会减低,是一种安全有效的扩增体系。     

Myeloid differentiation of human cord blood CD34+ cells during ex vivo expansion using thrombopoietin, flt3-ligand and/or granulocyte-colony stimulating factor

We investigated the phenotypic changes of human umbilical cord blood (CB) CD34+ cells during ex vivo expansion using thrombopoietin (TPO), flt3-ligand (FL), and/or granulocyte-colony stimulating factor (G-CSF). During ex vivo expansion of CD34+ cells isolated from human CB for up to 5 weeks, surface expression of molecules on the cultured cells including CD64 (Fc gammaRI), CD32 (Fc gammaRII), CD16 (Fc gammaRII), CD11b (MAC-1) and CD18 (beta2-integrin) was analysed by flow cytometry along with simultaneous measurement of apoptosis by 7-aminoactinomycin D staining method. CD64, CD32 and/or CD18 expressing cells appeared in the cultures both with and without the addition of G-CSF until the tenth day. However, without G-CSF, CD16+ fractions did not appear and CD11b+ fractions were not maintained. With G-CSF, the CD16+ or CD11b+ fractions appeared only from the second week. These results suggest that G-CSF is necessary for the late stage of myeloid maturation during which CD16 and CD11b are expressed.     

Ex vivo expansion of mature human neutrophils with normal functions from purified peripheral blood CD34+ haematopoietic progenitor cells

Purified CD34+ haematopoietic progenitor cells were cultivated with stem cell factor, interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte CSF (G-CSF) for 7 d, and thereafter non-adherent cells were divided into two groups. Cells in one group (group A) were further cultivated for 7 d with four cytokines, and cells in the other group (group B) were further cultivated for 7 d with G-CSF alone. On day 14, 220-fold and 130-fold increases in the numbers of non-adherent cells were achieved for groups A and B respectively. These cell preparations contained 65% granulocytes for group A and 95% granulocytes for group B. These cells gained the ability to respond effectively with chemotaxis, phagocytosis and superoxide (O2-) release. Cells in group B were appropriately primed by G-CSF, GM-CSF, tumour necrosis factor alpha and IL-1beta for enhanced release of O2 -. The responsiveness of these cells was identical to that of peripheral blood neutrophils, indicating that cells in group B may be in the resting state. In contrast, cells in group A were not primed by these cytokines for enhanced release of O2- and released a large amount of O2- spontaneously, indicating that cells in group A may be in the activated state. These findings indicate that mature neutrophils with normal functions were expanded ex vivo in group B and suggest that these cells could be used for possible autologous neutrophil transfusion to prevent bacterial infections during severe neutropenia after cytotoxic chemotherapy.     

The plant mannose-binding lectin NTL preserves cord blood haematopoietic stem/progenitor cells in long-term culture and enhances their ex vivo expansion

Ex vivo expansion of haematopoietic stem and progenitor cells in cytokine combinations is effective in promoting differentiation and proliferation of multilineage progenitor cells, but often results in reduction of self-renewable stem cells. This study investigated the effect of a mannose-binding lectin, NTL, purified from Narcissus tazetta var. chinensis , on prolonged maintenance and expansion of cord blood CD34⁺ cells. Our results showed that the presence of NTL or Flt-3 ligand (FL) significantly preserved a population of early stem/progenitor cells in a serum- and cytokine-free culture for 35 d. The effect of NTL on the ex vivo expansion of CD34⁺ cells in the presence of stem cell factor, thrombopoietin (TPO) and FL was also investigated. NTL-enhanced expansion of early progenitors (CD34⁺, CD34⁺CD38⁻, mixed colony-forming units and CFU-GEMM) and committed progenitor cells (granulocyte CFU, erythroid burst-forming units/CFU and megakayocyte CFU) after 8 and 12 d of culture. Six weeks after transplanting 12 d-expanded cells to non-obese diabetic severe combined immunodeficient mice, increased engraftment of human CD45⁺ cells was observed in the bone marrow of animals that received NTL-treated cells. The dual functions of NTL on long-term preservation and expansion of early stem/multilineage progenitor cells could be developed for applications in various cell therapy strategies, such as the clinical expansion of CD34⁺ cells for transplantation.     

Ex vivo expanded mobilized peripheral blood CD34+ cells accelerate haematological recovery in a baboon model of autologous transplantation

To address the value of ex vivo expanded haematopoietic cells for shortening cytopenia in autologous haematopoietic transplantation, we designed an ex vivo expansion protocol based on a cocktail of early acting cytokines and short-term culture and tested it in a baboon model. Expansion involved enriched CD34+ peripheral blood haematopoietic cells cultured for 6 d with a combination of FLT3-L, stem cell factor (SCF), thrombopoietin (TPO) and interleukin (IL)-3 (50 ng/ml each); CD34+ cells, granulocyte-macrophage colony-forming units (GM-CFU) and megakaryocytic colony-forming units (MK-CFU) were amplified, respectively, 10.5-, 20.5- and 17.9-fold. Baboons were submitted to a myeloablative regimen consisting of cyclophosphamide plus total body irradiation (TBI; 6 Gy) and were then grafted with either 2 x 106/kg unmanipulated CD34+ cells (control group, n = 4) or cells cultured from 2 x 106/kg CD34+ cells (expansion group, n = 4). No cytokines were administered after transplantation. All the animals engrafted. The mean times to white blood cell (WBC), granulocyte and platelet recovery were significantly shorter in the expansion group than in the control group: WBC (> 1 x 109/l) and neutrophil (> 0.5 x 109/l) recovery occurred on days 8 (range 6-9) and 9 (range 6-11), respectively, compared with days 12 (range 10-15) and 14 (range 11-16); platelets recovered (> 20 x 109/l) on day 9 (range 7-12) compared with day 13 (range 11-15) in the control group (P < 0.05). No toxicity was observed after reinfusion. No secondary hypoplasia was observed during more than 12 months of follow-up. Functions of both neutrophils and platelets produced from expanded cells were normal in terms of oxidative metabolism, chemotaxis and the bleeding time. This study shows that in comparison with unmanipulated cells peripheral blood haematopoietic cells expanded from similar doses of CD34+ cells, under the conditions defined here, accelerated both neutrophil and platelet recovery without impairing long-term haematopoiesis.     

Effects of thrombopoietin on the proliferation and differentiation of primitive and mature haemopoietic progenitor cells in cord blood

Thrombopoietin (TPO) is considered to be the primary growth factor for regulating megakaryopoiesis and thrombopoiesis. In this study we investigated the in vitro effect of TPO on relatively immature and mature CD34⁺ progenitor cells in cord blood. Cells were cultured in both liquid and semi-solid cultures containing 50 ng/ml TPO. The CD34⁺/CD45RA⁻ and CD34⁺/CD38⁻ subfractions in cord blood were both enriched for megakaryocyte progenitors as determined in a semisolid CFU-meg assay. Progenitor cells derived from the CD34⁺/CD45RA⁻ and CD34⁺/CD38⁻ subfractions showed high proliferative capacity in liquid cultures. We observed a mean 19-fold expansion of the total CD34⁺ cell fraction, whereas in the CD34⁺/CD45RA⁻ and CD34⁺/CD38⁻ subfractions the mean expansion was 23- and 50-fold respectively. The expansion of the immature progenitor cell subfractions resulted in a highly purified megakaryocyte suspension containing > 80% megakaryocytes after 14 d in culture. However, these expanded megakaryocytes remained in a diploid (2N) and tetraploid (4N) state. Maturation could not be further induced by low concentration of TPO (0.1 ng/ml). The majority of the cells were 2N (80%) and 4N (15%) and only 5% of the cells had a ploidy of more than 4N. These results indicate that megakaryocyte progenitor cells in cord blood residing in the immature stem cell fraction exhibit a high proliferative capacity when cultured in the presence of TPO as the single growth factor, without maturation to hyperploid megakaryocytes.     

辛伐他汀对大鼠平滑肌祖细胞和内皮祖细胞分化的不同影响

目的：观察辛伐他汀对平滑肌祖细胞（SPC）和内皮祖细胞（EPC）分化的影响。方法：采用密度梯度离心法获取大鼠骨髓单个核细胞,将其接种在纤维连接素包被培养板,加入不同浓度辛伐他汀（0．01～10μmol／L）培养8d。采用平滑肌肌动蛋白免疫荧光染色鉴定骨髓源性SPC,激光共聚焦显微镜鉴定FITC—UEA—I和Di I-acLDL双染阳性细胞为正在分化的EPC,并在倒置荧光显微镜下计数。结果：辛伐他汀显著抑制骨髓单个核细胞分化为SPC。0．01μmol／L辛伐他汀组与对照组SPC数量分别为79±5对85±4（P〈0．05）。辛伐他汀显著促进骨髓单个核细胞向EPC分化,其促进作用随辛伐他汀浓度升高而增加,在1．0μmol／L达最大效应。1μmol／L辛伐他汀组与对照组EPC数量分别为87±5对39±4（P〈0．01）。结论：辛伐他汀选择性抑制骨髓单个核细胞向SPC分化,促进其向EPC分化,局部应用有促进损伤血管再内皮化和抑制新生内膜过度增生的可能。     

胚胎干细胞体外定向分化为Ngn3+胰岛祖细胞的基因表达谱分析

目的 探讨胚胎干细胞体外定向分化为胰岛祖细胞的基因表达谱.方法 采用逐步诱导分化方案体外传代培养胚胎干细胞,应用RT-PCR和免疫细胞化学方法鉴定各分化阶段相关特异基因的表达,应用Illumina Mouse Ref-8 vl.1小鼠基因表达谱芯片测定胚胎干细胞来源的不同阶段的分化细胞(第4、8、15、20、22和25天)与未分化胚胎干细胞的基因组表达谱.结果 本研究得到了86个阶段特异性表达基因,6组具有相同表达趋势的基因簇,6组阶段差异表达基因组.从数量上看,差异表达基因最多的阶段是后前肠阶段(201个基因),接下来是定形内胚层细胞阶段(17个基因).结论 应用基因芯片对胰腺发育过程中具有相同表达趋势的基因和阶段差异表达基因的分析为早期胚胎发育和胰腺发育研究提供了试验依据.