Interleukin 3 improves the ex vivo expansion of primitive human cord blood progenitor cells and maintains the engraftment potential of scid repopulating cells.

In umbilical cord blood (UCB) transplantation, the number of nucleated cells per kilogram is a major predictive and critical factor of hematopoietic recovery. Thus, ex vivo expansion of hematopoietic UCB progenitors could potentially accelerate engraftment. Whereas Flt-3 ligand (FL), stem cell factor (SCF), and thrombopoietin (TPO) are considered indispensable, the role of interleukin 3 (IL-3) is still controversial: it has been reported either to support or abrogate the reconstituting ability of stem cells. By adding IL-3 we aimed to enhance the amplification of early and committed progenitor cells without impairing the long-term engraftment of stem cells. Demonstrating a positive impact of IL-3 on the proliferation of all progenitor subsets, the amplification of CD34+ UCB cells was increased 20.9-fold +/- 5.4 (mean +/- standard error) in serum-free culture with FL, SCF, TPO, and IL-3 as opposed to 9.3-fold +/- 3.2 without IL-3 after 7 days. If IL-3 was included, primitive long-term culture-initiating cells and committed colony-forming cells were expanded 16.3-fold +/- 5.5 and 18.1-fold +/- 2.4, respectively, compared to 12.6-fold +/- 5.6 and 9.1-fold +/- 2.0 without IL-3. Analysis of cultured CD34+ UCB cells in sublethally irradiated nonobese diabetic/severe combined immunodeficient mice confirmed that cultured cells had preserved their repopulating potential. After 6 weeks, all mice showed multilineage engraftment with their bone marrow containing an average of 45% human CD45+ cells of the unmanipulated sample, 43% of cells after culture in the presence of IL-3, and 27% of cells after culture without IL-3. In combination with early acting cytokines, IL-3 therefore improves the ex vivo expansion of UCB stem and progenitor cells without impairing their engraftment potential.     

Karyotyping, immunophenotyping, and apoptosis analyses on human hematopoietic precursor cells derived from umbilical cord blood following long-term ex vivo expansion

By means of flow cytometry, CD34+/CD38- hematopoietic stem cells (HSC) were collected from umbilical cord blood (UCB) of 10 healthy women at the time of delivery and cultivated in stem-cell culture media supplemented with cell growth stimulating factors (IL-3, IL-6, GM-CSF, EPO, IGF-1, and SCF) for long periods. Apoptotic status, cell surface marker expression, and karyotypes of the cultured UCB-derived CD34+/CD38- stem-cells were investigated by flow cytometry and GTG-banding methods. The UCB-derived CD34+/CD38- stem-cells were able to divide and proliferate in vitro for at least 6 months. They did not show significantly increased apoptosis following ex vivo expansion for 20 and 32 days, respectively, in 2 cases and retained the same cell surface marker expression pattern (i.e., CD34+ and CD38-) in the majority of the cells of 2 cases following 20 and 37 days of incubation, respectively. In another 2 cases, chromosome analysis showed no evidence of numerical and structural abnormalities in the CD34+/CD38- stem-cells obtained after 20 and 43 days in culture, respectively. Our findings indicated that UCB-derived CD34+/CD38- stemcells are able to maintain their basic biologic and genetic characteristics after dividing and proliferating in vitro for a long period of time. UCB-derived HSC following ex vivo expansion can serve as a reliable resource for hematopoietic precursor cells transplantation.     

LPAM-1 (integrin α4β7)-ligand binding: overlapping binding sites recognizing VCAM-1, MAdCAM-1 and CS-1 are blocked by fibrinogen,a fibronectin-like polymer and RGD-like cyclic peptides

The α4 integrin LPAM-1 (α4β7) mediates lymphocyte attachment within the extracellular matrix (ECM) by adhering to the connecting segment (CS)-1 site of fibronectin (FN). Here we reveal that very late antigen (VLA)-4⁻ LPAM-1⁺ T cell lymphoma TK-1 cells bind via LPAM-1 to multiple copies of the RGD sequence engineered within an FN-like polymer. Further, the small conformationally restrained RGD-like cyclic peptides 1-adamantaneacetyl-Cys-Gly-Arg-Gly-Asp-Ser-Pro-Cys and Arg-Cys-Asp-thioproline-Cys inhibit the adhesion of TK-1 cells to immobilized CS-1 peptide, and to endothelial counterreceptors for LPAM-1, namely mucosal addressin cell adhesion molecule (MAdCAM)-1 and vascular cell adhesion molecule (VCAM)-1. Spontaneous adhesion of the VLA-4⁻ LPAM-1⁺ B lymphoma cell line RPMI 8866 to CS-1 was likewise inhibited, confirming a previously undocumented ability of LPAM-1 to recognize the RGD tripeptide. The RGD-binding site in LPAM-1 either overlaps or is identical to sites required for interaction with MAdCAM-1, VCAM-1, and the CS-1. The binding of LPAM-1 and VLA-4 to RGD-containing ligands may have relevance in vivo given that fibrinogen at physiological concentrations is able to partially block the binding of TK-1 cells to MAdCAM-1. Hence fibrinogen and other vascular RGD-containing proteins may have mild anti-inflammatory activity required for maintaining effective homeostasis, analogous to the anti-thrombogenic activity of the vascular endothelium.     

Cell cycling status of human cord blood CD34+ cells during ex vivo expansion is related to the level of very late antigen expression

Very late antigen-4 (VLA-4), which binds to the extracellular matrix protein fibronectin, is an integrin molecule known to be modulated during mobilization of CD34+ cells, and to be involved in signaling the mobilization stimuli. On the hypothesis that cell cycling status might be different depending on the level of VLA-4 expression, we investigated the DNA contents of human cord blood CD34+ cells during ex vivo expansion by recombinant human thrombopoietin and flt3-ligand with simultaneous measurement of surface VLA-4 at the 1st and 4th week. During this ex vivo expansion, expression of VLA-4 increased and almost all cells became VLA-4+ until the 4th day of culture. Expression of VLA-4 was maintained in the major population of the cultured cells until the 4th week. The cells in S/G2/M phase were greater in number in VLA-4 high fraction than in VLA-4 low fraction (n=4, p<.001). Furthermore, the fraction of cells in S/G2/M phase increased as the expression of VLA-4 became higher. These results suggest that cord blood CD34+ cells expressing high levels of VLA-4 have more proliferative activities.     

Phenotypic and functional reversal within the early human hematopoietic compartment

The fate of phenotypically defined human hematopoietic stem cells (hHSCs) in culture and the link between their surface marker expression profile and function are still controversial. We studied these aspects of hHSC biology by relating the expression of the early lineage markers (ELM) CD33, CD38, and CD71 on the surface of human umbilical cord blood (UCB) CD34(+) cells to their long-term nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse repopulation activity (LT-SRA). In uncultured UCB samples, LT-SRA was largely confined to the small CD34(+)ELM(-) cell fraction. CD34(+) cells expressing ELM markers at their surface usually lacked LT-SRA. After culturing UCB CD34(+) cells for 6 days in serum-free medium and on a feeder layer of Rat2 cells, the number of CD34(+)ELM(-) cells stayed roughly the same or showed a slight increase and the LT-SRA was preserved, suggesting a close association between LT-SRA and the CD34(+)ELM(-) phenotype. Indeed, transplantation of CD34(+)ELM(-) cells isolated from cultured UCB CD34(+) cells resulted in long-term hematopoietic reconstitution of conditioned NOD/SCID mice, whereas CD34(+)ELM(+) cells derived from the same cultures were devoid of LT-SRA. Remarkably, roughly 1% of the cells recovered from cultures initiated with isolated CD34(+)ELM(+) cells had lost ELM surface expression. Concurrently, the cultured CD34(+)ELM(+) cells acquired LT-SRA, suggesting that hematopoietic stem cells (HSCs) may arise by the dedifferentiation of early hematopoietic progenitor cells. The latter finding challenges the paradigm of unidirectional hematopoietic differentiation and opens new opportunities for HSC expansion prior to transplantation.     

Characterization of a lineage-negative stem-progenitor cell population optimized for ex vivo expansion and enriched for LTC-IC

Current hematopoietic stem cell transplantation protocols rely heavily upon CD34+ cells to estimate hematopoietic stem and progenitor cell (HSPC) yield. We and others previously reported CD133+ cells to represent a more primitive cell population than their CD34+ counterparts. However, both CD34+ and CD133+ cells still encompass cells at various stages of maturation, possibly impairing long-term marrow engraftment. Recent studies demonstrated that cells lacking CD34 and hematopoietic lineage markers have the potential of reconstituting long-term in vivo hematopoiesis. We report here an optimized, rapid negative-isolation method that depletes umbilical cord blood (UCB) mononucleated cells (MNC) from cells expressing hematopoietic markers (CD45, glycophorin-A, CD38, CD7, CD33, CD56, CD16, CD3, and CD2) and isolates a discrete lineage-negative (Lin-) cell population (0.10% +/- 0.02% MNC, n=12). This primitive Lin- cell population encompassed CD34+/- and CD133+/- HSPC and was also enriched for surface markers involved in HSPC migration, adhesion, and homing to the bone marrow (CD164, CD162, and CXCR4). Moreover, our depletion method resulted in Lin- cells being highly enriched for long-term culture-initiating cells when compared with both CD133+ cells and MNC. Furthermore, over 8 weeks in liquid culture stimulated by a cytokine cocktail optimized for HSPC expansion, TPOFLK (thrombopoietin 10 ng/ml, Flt3 ligand 50 ng/ml, c-Kit ligand 20 ng/ml) Lin- cells underwent slow proliferation but maintained/expanded more primitive HSPC than CD133+ cells. Therefore, our Lin- stem cell offers a promising alternative to current HSPC selection methods. Additionally, this work provides an optimized and well-characterized cell population for expansion of UCB for a wider therapeutic potential, including adult stem cell transplantation.     

Ex vivo expansion of stem cells from umbilical cord blood: expression of cell adhesion molecules

Expansion of stem cells from cord blood has been demonstrated to increase the numbers of CD34+ cells, CD34+ subsets, long-term culture-initiating cells, and severe combined immunodeficient mouse, repopulating cells. However, reports suggest that the ex vivo expanded population behaves differently than freshly isolated cells and shows a delayed or diminished engraftment. In this study, we investigated the effects of the cytokines flt3 ligand, stem cell factor, and thrombopoietin on expansion of CD34+ and CD34+/CD38- cells. In addition, we studied the expression of adhesion molecules, very late activation antigen-4 (VLA-4) and leukocyte function antigen-1 (LFA-1), on CD34+ cells from cord blood by flow cytometry. We also looked at the expression of an adhesion receptor, namely, vascular cell adhesion molecule-1 (VCAM-1) on bone marrow stromal cells by Western blot analysis after exposure to low dose gamma irradiation. After culturing for 7 days, increases in the absolute numbers of CD34+, CD34+/CD38-, CD34+/VLA-4+, and CD34+/LFA-1+ cells were 5.67 +/- 2.91 (mean +/- standard deviation) fold, 7.21 +/- 4.38 fold, 99.56 +/- 101.5 fold, and 101.39 +/- 83.25 fold, respectively. There was a transient upregulation in the expression levels of VCAM-1 on stromal cells, which peaked at 4 hours. Though there was an increase in the absolute numbers of CD34+ cells expressing the adhesion molecules, the expression levels (antigen density) of the adhesion molecules on the CD34+ cells remained unaffected.     

RGD肽对内皮细胞在聚酯材料表面粘附、增殖的影响

RGD是许多粘附蛋白结构中的高度保守序列，与细胞在生物材料表面的粘附、增殖密切相关。本研究在聚酯薄膜表面分别预衬纤维粘连蛋白和共价接枝RGD三肽，然后在不同聚酯材料上种植体外培养的人脐静脉内皮细胞，结果显示RGD可明显促进细胞在材料表面的粘附和增殖，与纤维粘连蛋白相比，RGD促进细胞粘附的作用更为明显，而在细胞增殖方面，二者的作用无显著性差异。本研究为改进生物材料的表面设计，促进心血管移植物的内皮化提供了一个切实可行的思路。     

成人骨髓间充质干细胞对造血干细胞体外增殖的影响

目的研究骨髓间充质干细胞（MSC）对脐带血（CB）CD34^＋细胞体外增殖和造血重建能力的影响。方法取人骨髓单个核细胞贴壁培养．梭形细胞完全融合后传代，用流式细胞仪检测免疫表型；将CBCD34^＋细胞接种到MSC或其他培养液中．比较不同培养条件对造血干细胞扩增能力、集落形成能力及黏附分子表达的影响。结果在加入IL-3的培养体系中．在MSC和细胞因子作用下，CD34^＋细胞扩增7d和14d后，有核细胞（NC）、CD34^＋细胞和CDl33^＋细胞数，实验组均显著多于对照组。CD34＋细胞在未加入IL-3的培养体系中培养8d后，实验组NC、CD34^＋细胞、CD34^＋CD38-细胞和造血祖细胞集落扩增倍数均显著高于对照组。扩增后CD34^＋细胞的ALCAM、VLA-α4、VLA-α5、VLA-β1、HCAM、PECAM和LFA-1表达较扩增前无显著变化。结论MSC可为造血干细胞（HSC）体外扩增提供适宜的微环境，有助于CD34^＋细胞体外增殖并抑制HSC分化，保持其造血重建潜能和归巢能力。     

Stem cells expressing homing receptors could be expanded from cryopreserved and unselected cord blood

We assessed the cytokine combinations that are best for ex vivo expansion of cord blood (CB) and the increment for cell numbers of nucleated cells, as well as stem cells expressing homing receptors, by an ex vivo expansion of cryopreserved and unselected CB. Frozen leukocyte concentrates (LC) from CB were thawed and cultured at a concentration of 1 x 10(5)/mL in media supplemented with a combination of SCF (20 ng/mL)+TPO (50 ng/mL)+FL (50 ng/mL)+/-IL-6 (20 ng/mL)+/-G-CSF (20 ng/mL). After culturing for 14 days, the expansion folds of cell numbers were as follows: TNC 22.3+/-7.8 approximately 26.3+/-4.9, CFU-GM 4.7+/-5.1 approximately 11.7+/-2.6, CD34+CD38- cell 214.0+/-251.9 approximately 464.1+/-566.1, CD34+CXCR4+ cell 4384.5+/-1664.7 approximately 7087.2+/-4669.3, CD34+VLA4+ cell 1444.3+/-1264.0 approximately 2074.9+/-1537.0, CD34+VLA5+ cell 86.2+/-50.9 approximately 113.2+/-57.1. These results revealed that the number of stem cells expressing homing receptors could be increased by an ex vivo expansion of cryopreserved and unselected CB using 3 cytokines (SCF, TPO, FL) only. Further in vivo studies regarding the engraftment after expansion of the nucleated cells, as well as the stem cells expressing homing receptors will be required.     

Ex vivo expansion of megakaryocyte precursors from umbilical cord blood CD34 cells in a closed liquid culture system.

Umbilical cord blood (UCB) provides a rich source of stem cells for transplantation after myeloablative therapy. One major disadvantage of UCB transplantation is delayed platelet engraftment. We propose to hasten platelet engraftment by expanding the number of megakaryocyte (MK) precursors (CD34/CD41 cells) through cytokine stimulation within a closed, pre-clinical liquid culture system. Clinical engraftment data suggest a 5- to 10-fold increase in MK precursors in a UCB unit can accelerate platelet engraftment, so this was our goal. Thirteen UCB samples from full-term births were Ficoll-separated and frozen for subsequent use. On thawing, the mononuclear cell population was positively selected for CD34(+) expression. The cells were cultured in gas-permeable Teflon-coated bags in serum-free medium containing the following cytokines: recombinant human interleukin-3, recombinant human Flt3 ligand, recombinant human stem cell factor, and recombinant human thrombopoietin. MK lineage cell expansion was assessed using mononuclear cell count and flow cytometry (CD34/41, CD41, CD34/61, and CD61 expression) on days 7, 11, and 14. Optimal expansion of CD34/41 and CD41 cells was observed at day 11, with a median 6-fold and 33-fold increase in the starting cell doses, respectively. CD34/61 and CD61 cell expansion at day 11 was 7-fold and 14-fold, respectively. MK precursors can be successfully expanded from CD34(+) UCB cells in a closed liquid culture system using interleukin-3, recombinant human Flt3 ligand, recombinant human stem cell factor, and recombinant human thrombopoietin to a level that should have a clinical impact in the transplantation setting. Our ex vivo expansion technique needs to be further optimized before it can be used in a pilot UCB transplantation trial.     

Expression of adhesion molecules on CD34(+) cells in peripheral blood of non-hodgkin's lymphoma patients mobilized with different growth factors

Adhesion molecules on CD34(+) cells were implicated in the process of peripheral blood stem cell (PBSC) mobilization and homing. We studied the mobilization of CD34(+)Thy1(+) cells, CD34(+) very late-acting antigen (VLA)4(+) cells, and CD34(+)L-selectin(+) cells in non-Hodgkin's lymphoma patients mobilized with cyclophosphamide plus G-CSF, GM-CSF, or GM-CSF followed by G-CSF. The mean percentage of CD34(+) cells in the bone marrow (BM) expressing Thy1 was 23.6% +/- 11% and 17.8% +/- 8% in the PB before mobilization, and was markedly decreased to 4.5% +/- 3.3% in the apheresis collections. Similarly, the mean percentage of CD34(+) cells expressing L-selectin was 35.8% +/- 4.3% in the BM, 21.6% +/- 4.1% in the PB before mobilization and was markedly decreased to 9.1% +/- 2.5% in the apheresis collections. Patients in the three arms of the study had a similar pattern of CD34(+)Thy1(+) and CD34(+)L-selectin(+) cell mobilization. Also, a similar pattern of coexpression of CD34(+)Thy1(+) and CD34(+)L-selectin(+) cells was observed when the patients were regrouped as "good mobilizers" (> or =2 x 10(6) CD34(+)CD45(dim) cells/kg, in four collections) and "poor mobilizers" (<0.4 x 10(6) CD34(+)CD45(dim) cells/kg, in two collections). The mean percentage of CD34(+) cells expressing VLA-4 in the BM and PB was relatively high (73.4% +/- 12% and 65.4% +/- 6.6%, respectively) and dropped considerably in the PBSC collections to 43.5% +/- 7.1% with a similar pattern observed for patients in arms A, B, and C. However, when the patients were regrouped as "good mobilizers" and "poor mobilizers," a higher percentage of CD34(+) cells expressing VLA-4 was observed in the PBSC of the pooled "good mobilizers" (50.5% +/- 9% versus 36.3% +/- 6.4%; p = 0.01). We conclude that release of CD34(+) cells to the PB involves a general downregulation of Thy1, L-selectin and VLA-4 on CD34(+) cells, irrespective of the growth factor used for mobilization. However, good mobilizers had a relatively higher percentage of CD34(+) cells expressing the VLA-4 antigen.     

Acrylic Acid-RGD as a Biomimetic Surface Modifier for Poly(ethylene terephthalate)

INTRODUCTION　Biomaterials play an importantrole in human disease- treatmentand healing〔1,2〕.Due to the good mechanical property,PET is used to the coating of artificial heartvalve,the film of mending hearts and artificial vessel etc〔3〕.But the imperfection isthe low capability of surface hydrophile leading to the high static and low water ad-sorption〔4〕.In the application,traditional artificial cardiovascular materials( e.g.PET) have blood coagulation,alexin- activation and other…     

Expansion of LTC-ICs and maintenance of p21 and BCL-2 expression in cord blood CD34(+)/CD38(-) early progenitors cultured over human MSCs as a feeder layer

Allogeneic transplantation with umbilical cord blood (UCB) is limited in adult recipients by a low CD34(+) cell dose. Clinical trials incorporating cytokine-based UCB in vitro expansion have not demonstrated significant shortening of hematologic recovery despite substantial increases in CD34(+) cell dose, suggesting loss of stem cell function. To sustain stem cell function during cytokine-based in vitro expansion, a feeder layer of human mesenchymal stem cells (MSCs) was incorporated in an attempt to mimic the stem cell niche in the marrow microenvironment. UCB expansion on MSCs resulted in a 7.7-fold increase in total LTC-IC output and a 3.8-fold increase of total early CD34(+) progenitors (CD38(-)/HLA-DR(-)). Importantly, early CD34(+)/CD38(-)/HLA-DR(-) progenitors from cultures expanded on MSCs demonstrated higher cytoplasmic expression of the cell-cycle inhibitor, p21(cip1/waf1), and the antiapoptotic protein, BCL-2, compared with UCB expanded in cytokines alone, suggesting improved maintenance of stem cell function in the presence of MSCs. Moreover, the presence of MSCs did not elicit UCB lymphocyte activation. Taken together, these results strongly suggest that the addition of MSCs as a feeder layer provides improved conditions for expansion of early UCB CD34(+)/CD38(-)/HLA-DR(-) hematopoietic progenitors and may serve to inhibit their differentiation and rates of apoptosis during short-term in vitro expansion.     

Cytokine expansion culture of cord blood CD34+ cells induces marked and sustained changes in adhesion receptor and CXCR4 expressions

Recent studies have demonstrated defective bone marrow homing of hematopoietic stem cells after cytokine expansion culture. Adhesion receptors (ARs) are essential to the homing process, and it is possible that cytokine culture modulates AR expression. We studied changes in expression of very late antigen-4 (VLA-4), VLA-5, L-selectin, leukocyte function-associated antigen-1 (LFA-1), CD44, and the stromal cell-derived factor-1 (SDF-1) receptor, CXCR4, during cytokine culture of cord blood (CB) CD34(+) cells. Expression of ARs was studied by flow cytometry on CB CD34(+) cells in whole blood, after purification and during culture for up to 10 days. Cells were cultured with stem cell factor (SCF), thrombopoietin (TPO), Flt3-ligand (Flt3), and G-CSF. Results showed that 80% or more of uncultured CD34(+) cells were positive for VLA-4, L-selectin, LFA-1, CD44, and CXCR4 while 50% were positive for VLA-5. Purification of CD34(+) cells did not affect AR expression, but cytokines increased expression three- to nine-fold throughout the 10-day culture period. In contrast, expression of CXCR4 decreased. Expression changes of ARs and CXCR4 on CD34(+)/CD38(-) cells mirrored those of the total CD34(+) population. The results indicate that cytokine culture significantly increases AR expression on CB CD34(+) cells, which may be related to the decrease in homing of cytokine-cultured hematopoietic stem cells.     

Adherent cells generated during long-term culture of human umbilical cord blood CD34+ cells have characteristics of endothelial cells and beneficial effect on cord blood ex vivo expansion

Hematopoiesis depends on the association of hematopoietic stem cells with stromal cells that constitute the hematopoietic microenvironment. The in vitro development of the endothelial cell from umbilical cord blood (UCB) is not well established and has met very limited success. In this study, UCB CD34(+) cells were cultured for 5 weeks in a stroma-free liquid culture system using thrombopoietin, flt3 ligand, and granulocyte-colony stimulating factor. By week 4-5, we found that firmly adherent fibroblast-like cells were established. These cells showed characteristics of endothelial cells expressing von Willebrand factor, human vascular cell adhesion molecule-1, human intracellular adhesion molecule-1, human CD31, E-selectin, and human macrophage. Furthermore, when comparing an ex vivo system without an established endothelial monolayer to an ex vivo system with an established endothelial monolayer, better expansion of total nucleated cells, CD34(+) cells, and colony-forming units (CFUs)-granulocyte-macrophage and CFUs-granulocyte-erythroid-megakaryocyte-macrophage were found during culture. This phenomenon was in part due to the fact that a significant reduction of apoptotic fractions was found in the CD34(+) cells, which were cultured on the adherent monolayer for up to 5 weeks. To gather quantitative data on the number of endothelial cells derived from a given number of CD34 cells, we performed limiting dilution assay by using Poisson distribution: the number of tested cells (linear scale) producing a 37% negative culture (logarithmic scale) is the number of cells containing one endothelial cell. By this method, one endothelial cell may be found from 314 CD34(+) cells after 5 weeks of culture. These results suggest that the UCB CD34(+) cell fraction contains endothelial cell precursors, establishing the hematopoietic microenvironment and providing the beneficial effects through downregulating apoptosis on UCB expansion protocols. These observations may provide insight for future cellular therapy or graft engineering.     

In vitro expanded cells contributing to rapid severe combined immunodeficient repopulation activity are CD34+38-33+90+45RA-

Expansion of hematopoietic stem cells could be used clinically to shorten the prolonged aplastic phase after umbilical cord blood (UCB) transplantation. In this report, we investigated rapid severe combined immunodeficient (SCID) repopulating activity (rSRA) 2 weeks after transplantation of CD34(+) UCB cells cultured with serum on MS5 stromal cells and in serum- and stroma-free cultures. Various subpopulations obtained after culture were studied for rSRA. CD34(+) expansion cultures resulted in vast expansion of CD45(+) and CD34(+) cells. Independent of the culture method, only the CD34(+)33(+)38(-) fraction of the cultured cells contained rSRA. Subsequently, we subfractionated the CD34(+)38(-) fraction using stem cell markers CD45RA and CD90. In vitro differentiation cultures showed CD34(+) expansion in both CD45RA(-) and CD90(+) cultures, whereas little increase in CD34(+) cells was observed in both CD45RA(+) and CD90(-) cultures. By four-color flow cytometry, we could demonstrate that CD34(+)38(-)45RA(-) and CD34(+)38(-)90(+) cell populations were largely overlapping. Both populations were able to reconstitute SCID/nonobese diabetic mice at 2 weeks, indicating that these cells contained rSRA activity. In contrast, CD34(+)38(-)45RA(+) or CD34(+)38(-)90(-) cells contributed only marginally to rSRA. Similar results were obtained when cells were injected intrafemorally, suggesting that the lack of reconstitution was not due to homing defects. In conclusion, we show that after in vitro expansion, rSRA is mediated by CD34(+)38(-)90(+)45RA(-) cells. All other cell fractions have limited reconstitutive potential, mainly because the cells have lost stem cell activity rather than because of homing defects. These findings can be used clinically to assess the rSRA of cultured stem cells.     

Defining optimum conditions for the ex vivo expansion of human umbilical cord blood cells. Influences of progenitor enrichment, interference with feeder layers, early-acting cytokines and agitation of culture vessels

Ex vivo expansion of human umbilical cord blood cells (HUCBC) is explored by several investigators to enhance the repopulating potential of HUCBC. We performed experiments using either Ficoll-separated or CD34+-selected HUCBC from the same donation in serum-free medium. CD34-purified HUCBC were cultured on either human umbilical vein endothelial cells (HUVEC) or irradiated bone marrow-derived stroma cells (BMSC) with addition of different cytokines. In addition, we tested the expansion of HUCBC in culture vessels with continuous rotation. CD34 enrichment led to a significant increase in the expansion factor of CD34+ cells compared with unmanipulated HUCBC. BMSC were more efficient in amplifying early progenitors than HUVEC. Optimum results were reached by a combination of SCF, FLT-3L at 300 ng/ml and IL-3 at 50 ng/ml. No significant improvement in the expansion of CD34+/38- primitive progenitors could be obtained with other combinations. Addition of megakaryocyte-derived growth and development factor to each growth factor cocktail improved the expansion results. Continuous rotation of culture vessels did not ameliorate the expansion rate of the analyzed subsets. Culture conditions separating stroma and HUCBC by a semipermeable membrane improved the expansion factors of CD34+, CD34+/38-, and CD34+/41+ cells and CFU-GM compared with contact cultures. These data might be useful when designing culture systems for clinical scale ex vivo expansion of HUCBC.     

人胎盘CD133+细胞具有高增殖潜能集落形成细胞特性

目的 通过对人胎盘CD133⁺细胞群中高增殖潜能集落形成细胞(HPP-CFC)检测与生物学特性的分析，证明人胎盘存在早期造血干/祖细胞（HSPC）。 方法 采用机械法制备人胎盘组织（PT）单细胞悬液，用Histopaque-1007分离出单个核细胞(MNC)，经磁式分选（MACS）富集CD133⁺细胞，培养28 d后观察HPP-CFC集落形成能力，用流式细胞仪（FCM）对分选的细胞组份和HPP-CFC进行表型分析，实验全程用脐带血（UCB）作平行比较分析。 结果 培养28 d后，PT-CD133⁺与UCB-CD133⁺细胞组份分别扩增了266和362倍，前者低于后者（P<0.01）；PT-CD133⁺与UCB-CD133⁺细胞中HPP-CFC分别为(32.4±11.2)/5×10³、(17. 7±5.7)/5×10³，前者形成的HPP-CFC数量明显高于后者（P<0.01）；PT-CD133⁺、UCB-CD133⁺细胞培养至28 d时，除UCB-CD133⁺组的CD133⁺CD34^-亚群比例无明显改变外，CD133⁺CD34⁺、CD133^-CD34⁺和CD133⁺CD34^-（PT-CD133⁺组）亚型均比培养前减少。 结论 人胎盘组织CD133⁺细胞中存在HPP-CFC，说明胎盘CD133⁺细胞群中存在早期HSPC。     

Macrophage adhesion on gelatin-based interpenetrating networks grafted with PEGylated RGD

Human blood-derived macrophage adhesion on interpenetrating networks (IPNs) composed of PEGylated RGD-modified gelatin and poly(ethylene glycol) diacrylate was studied. The interaction between biomaterial immobilized with biofunctional peptides such as RGD and macrophages is central in the design of tissue-engineering scaffolds. PEGylated RGD-modified gelatin was synthesized via several steps involving PEG derivations and characterized by high-performance liquid chromatography, mass spectroscopy, gel permeation chromatography, and the trinitrobenzenesulfonic acid method. IPNs containing modified or unmodified gelatin were cultured with human macrophages and monitored at 2, 24, 96, and 168 h. At each time point, IPNs containing gelatin modified with PEGylated RGD showed a comparable adherent macrophage density as tissue culture polystyrene and a significantly higher cell density than other IPN formulations containing unmodified gelatin or gelatin modified with PEGylated triglycine. Although surface-immobilized RGD can serve to mediate the adhesion of different cell types on the biomaterial surface, the interaction of RGD with immune/inflammatory cells such as macrophages should also be considered when assessing the potential host response of tissue-engineering scaffolds.