Culture Of Cord Blood CD34 Research Articles

Generation of Megakaryocytes and Platelets from Valproic Acid Expanded Human Cord Blood CD34+ Cells in Culture

Currently in the US alone there are more than 2 million units of platelets transfused each year. The transfusion of platelets can be crucial life saving measures for patients undergoing treatment for leukemia and other cancers. However, volunteer donors are the only source of platelets for transfusions. The objectives of our studies are to further refine our multi-step culture strategies to expand human umbilical cord blood (CB) derived megakaryocyte (MK) progenitors, and augment functional MK/platelet yields. We used serum-free culture to expand stem/progenitor cells over 5 to 7 days from CB and growth factor mobilized peripheral blood (mPB) CD34+ cells using valproic acid (VPA), a histone deacetylase inhibitor (HDAC). VPA-expanded CB cells were assayed for MK progenitors by flow cytometry and CFU-MK assays. Compared to mPB, CB CD34+ cells yielded relatively more CD34+CD90+ cells (>1.6 fold), yet the absolute numbers of CD34+CD41a+ MK progenitors were comparable (CB 7.18 ± 1.19 x 105/well vs. mPB 7.55 ± 1.14 x 105/well). The CFU-MK colony yield from mPB was relatively lower than CB (CB 32.31 ± 9.57 x 103/well vs. mPB 11.3 ± 5.74 x 103/well, not significant). Extending the CB cultures up to 7 days resulted in notably greater fold increase of CD34+CD90+ primitive stem/progenitor cells from prior to expansion (D5 VPA 75.77 ± 14.83 vs. D7 VPA 146.26 ± 28.52-fold increase of CD34+CD90+ cells; p = 0.07, n = 4) as well as increase in CD34+CD41a+ MKs (D5 VPA 1.02 ± 0.24 X 105/well, n = 8, vs. D7 VPA 2.29 ± 0.85 X 105/well, n= 6, p = 0.04) and slightly higher number of CFU-MK colonies. We then estimated CD41a+ MK cell yield per initial single CD34+ CB cell plated to initiate expansion culture. VPA-expanded CD34+ cells were further cultured in the presence of various small molecules including stem reginin 1 (SR1), UM171, VPA, and nicotinamide. The yield of CD41a+ MKs from a single CD34+ cell plated increased with time. Having UM171 or VPA in extended cultures is favorable to generate high yield CD41+ MKs. Interestingly, a single CD34+ cell following VPA-expansion yielded up to 2,060.32 ± 631.48 (mean ± SE, n= 6) CD41a+ MKs after 17 days of culture. If we plated every single cell from a CB unit containing 5 million CD34+ cells in VPA expansion culture and continued the cultures for 17 days, 10.3 billion CD41a+ cells (10.3 x 109) could be generated. If we are capable of producing about 50 platelets per MK plated using optimized culture conditions, this would yield almost 2 units of transfusible platelets (5.15 x 1011) for patients. Using cultures of CB CD34+ cells, the relative expression levels of 12 genes were assessed by real-time qRT-PCR. Genes tested were classified according to the relative abundance of transcripts for the erythroid (EPOR and KLF1 ) and MK (AML1 , FLI1 , GABPA , and MPL ) lineages, as well as genes common to both lineages (GATA1 , NFE2 , TAL1 , GATA2 , MYB , and LMO2 ). Interestingly in MK inductive culture, VPA treated D14 samples displayed relatively lower transcript levels of erythroid genes including EPOR and KLF1 and higher expression of MK specific gene Fli1. After further differentiation at day 20 the expression of MK specific gene FLI1 achieved the highest level of expression (D0 to D7 1.84 ± 0.3; D0 to D20 3.52 ± 0.79-fold increase) and erythroid gene KLF1 had the lowest level of expression (D0 to D7 12.15 ± 1.72; D0 to D20 5.76 ± 3.77-fold increase) relative to pre-culture samples. Taken together, MK specific gene transcript levels increased while erythroid specific gene expression decreased following extended culture of CD34+ cells. These results suggest that VPA expanded CB cells maintain MK lineage commitment during extended culture. The MKs generated in culture appear to be relatively immature and only 21.44 ± 2.99 % (n = 4) of CD41a+ MKs co-expressed CD42b. However, in optimal MK condition the VPA expanded CB cells were capable of generating mature MKs (CD41a+/CD42b+) leading up to 3 to 7 times more CD41a+CD42b+ MKs in comparison to unexpanded CB CD34+ cells. Platelets generated per CD41a+CD42b+ MKs from VPA-expanded CD34+ cells were also 2 to 4 times greater than unexpanded CD34+ cells. Our current MK generation strategies from VPA-expanded CB cells, once further scaled up and optimized for MK maturation/platelet generation, has the potential to generate about two units of platelets for transfusion. DisclosuresNo relevant conflicts of interest to declare.

Human Umbilical Cord Blood CD34+Cells InducedAngiogenesis in Ischemic Limb of Mice.

Objective To observe the effect of the expanded human umbilical cord blood CD34+cells in ischemic limb of mice and analyse the relationship between the CD34+cells and angiogenesis. Methods Human umbilical cord blood was collected and CD34+cells were separated for expanding. Mice limbs ischemia models were established (n=15) and randomly divided into three groups:expanded CD34+cells group (n=5),fresh CD34+cells group (n=5),and control group(n=5). CD34+cells were detected by DiI dye tracing and antihuman nuclear antigen antibody(HNA) immunohistochemical staining. The improvement of blood reperfusion was evaluated by indicators including limb temperature,CD31 staining,and transforming growth factor-β1 (TGF-β1) mRNA expression. Results On days 14 (t=5.421,P=0.001;t=0.616,P=0.000) and 28(t=10.780,P=0.000; t=12.123,P=0.000),both expanded CD34+cells group and fresh CD34+cells group enjoyed better temperature improvement. Days 28 later,the vascular densities in the expanded CD34+cells group and the fresh CD34+cells group were 592.3±24.6 (t=26.386,P=0.000) and 530.7±25.5 (t=21.502,P=0.000),which were significantly higher than that in control group 219.7±19.9. The TGF-β1 mRNA expression in the expanded CD34+cells group and the fresh CD34+cells group were (0.578±0.050) copies (t=12.376,P=0.000) and (0.504±0.080) copies (t=7.098,P=0.000),both over control group [(0.224±0.040)copies]. Conclusions In vitro culture of cord blood CD34+cells can emigrate to ischemic zone and induce angiogenesis to alleviate ischemia. Thus,it may provide a treatment option for lower limb ischemia.

Definitive Setup of Clinical Scale Procedure for Ex Vivo Expansion of Cord Blood Hematopoietic Cells for Transplantation

We recently developed a clinical grade ex vivo cord blood expansion procedure enabling a massive amplification of hematopoietic progenitors without any loss of stem cell potential. This procedure, based on day 14 liquid cultures of cord blood CD34(+) cells, in medium Macopharma HP01 and in the presence of stem cell factor (SCF; 100 ng/ml), fms-related tyrosine kinase 3-ligand (Flt-3L; 100 ng/ml), megakaryocyte growth and developmental factor (MGDF; 100 ng/ml), and granulocyte colony-stimulating factor (G-CSF; 10 ng/ml) had to be modified due to the commercially unavailability of clinical grade MGDF molecule. So MGDF was replaced by thrombopoietin (TPO) in fivefold lower dose (20 ng/ml), and culture time was reduced to 12 days. That way, a mean expansion fold of 400, 80, and 150 was obtained for total cells, CD34(+) cells, and colony-forming cells (CFCs), respectively. This amplification was associated with a slight enhancing effect on stem cells [Scid repopulating cells (SRCs)]. These are the ultimate preclinical modifications of a clinical grade expansion protocol, which is already employed in an ongoing clinical trial.

Combination of low O2 concentration and mesenchymal stromal cells during culture of cord blood CD34+ cells improves the maintenance and proliferative capacity of hematopoietic stem cells

The physiological approach suggests that an environment associating the mesenchymal stromal cells (MSC) and low O(2) concentration would be most favorable for the maintenance of hematopoietic stem cells (HSCs) in course of ex vivo expansion of hematopoietic grafts. To test this hypothesis, we performed a co-culture of cord blood CD34(+) cells with or without MSC in presence of cytokines for 10 days at 20%, 5%, and 1.5% O(2) and assessed the impact on total cells, CD34(+) cells, committed progenitors (colony-forming cells-CFC) and stem cells activity (pre-CFC and Scid repopulating cells-SRC). Not surprisingly, the expansion of total cells, CD34(+) cells, and CFC was higher in co-culture and at 20% O(2) compared to simple culture and low O(2) concentrations, respectively. However, co-culture at low O(2) concentrations provided CD34(+) cell and CFC amplification similar to classical culture at 20% O(2) . Interestingly, low O(2) concentrations ensured a better pre-CFC and SRC preservation/expansion in co-culture. Indeed, SRC activity in co-culture at 1.5% O(2) was higher than in freshly isolated CD34(+) cells. Interleukin-6 production by MSC at physiologically low O(2) concentrations might be one of the factors mediating this effect. Our data demonstrate that association of co-culture and low O(2) concentration not only induces sufficient expansion of committed progenitors (with respect to the classical culture), but also ensures a better maintenance/expansion of hematopoietic stem cells (HSCs), pointing to the oxygenation as a physiological regulatory factor but also as a cell engineering tool.

Clinical-Scale Cultures of Cord Blood CD34+ Cells to Amplify Committed Progenitors and Maintain Stem Cell Activity

We developed a clinical-scale cord blood (CB) cell ex vivo procedure to enable an extensive expansion of committed progenitors--colony-forming cells (CFCs) without impairing very primitive hematopoietic stem cells (HSCs). CD34(++) cells, selected from previously cryopreserved and thawed CB units, were cultured in two steps (diluted 1:4 after 6 days) in the presence of stem cell factor (SCF), fms-related tyrosine kinase 3 ligand (Flt-3L), megakaryocyte growth and development factor (MGDF) (100 ng/ml each), granulocyte-colony stimulating factor (G-CSF) (10 ng/ml) in HP01 serum-free medium. HSC activity was evaluated in a serial transplantation assay, by detection of human cells (CD45, CD33, CD19 and CFC of human origin) in bone marrow (BM) of primary and secondary recipient NOD/SCID mice 6-8 weeks after transplantation. A wide amplification of total cells (∼350-fold), CD34(+) cells (∼100-fold), and CFC (∼130-fold) without impairing the HSC activity was obtained. The activity of a particular HSC subpopulation (SRC(CFC)) was even enhanced.Thus, an extensive ex vivo expansion of CFCs is feasible without impairing the activity of HSCs. This result was enabled by associating antioxidant power of medium with an appropriate cytokine cocktail (i.e., mimicking physiologic effects of a weak oxygenation in hematopoietic environment).

MicroRNA 223-dependent expression of LMO2 regulates normal erythropoiesis

MicroRNAs are small non-coding RNAs that regulate gene expression through mRNA degradation or translational inhibition. MicroRNAs are emerging as key regulators of normal hematopoiesis and hematologic malignancies. Several miRNAs are differentially expressed during hematopoiesis and their specific expression regulates key functional proteins involved in hematopoietic lineage differentiation. This study focused on the functional role of microRNA-223 (miR-223) on erythroid differentiation. Purified cord blood CD34+ hematopoietic progenitor cells were grown in strictly controlled conditions in the presence of saturating dosage of erythropoietin to selectively induce erythroid differentiation. The effects of enforced expression of miR-223 in unilin-eage erythroid cultures were evaluated in liquid phase culture experiments and clonogenic studies. In unilineage erythroid culture of cord blood CD34+ hematopoietic progenitor cells miR-223 is down-regulated, whereas LMO2, an essential protein for erythroid differentiation, is up-regulated. Functional studies showed that enforced expression of miR-223 reduces the mRNA and protein levels of LMO2, by binding to LMO2 3' UTR, and impairs differentiation of erythroid cells. Accordingly, knockdown of LMO2 by short interfering RNA mimics the action of miR-223. Furthermore, hematopoietic progenitor cells transduced with miR-223 showed a significant reduction of their erythroid clonogenic capacity, suggesting that downmodulation of this miRNA is required for erythroid progenitor recruitment and commitment. These results show that the decline of miR-223 is an important event for erythroid differentiation that leads to the expansion of erythroblast cells at least partially mediated by unblocking LMO2 protein expression.

Application of porous glycosaminoglycan‐based scaffolds for expansion of human cord blood stem cells in perfusion culture

In vitro expansion of hematopoietic stem cells (HSCs) has been employed to obtain sufficient numbers of stem cells for successful engraftment after HSC transplantation. A three-dimensional perfusion bioreactor system with a heparin-chitosan scaffold was designed and evaluated for its capability to support maintenance and expansion of HSCs. Porous chitosan scaffolds were fabricated by a freeze-drying technique and N-desulfated heparin was covalently immobilized within the scaffolds using carbodiimide chemistry. CD34+ HSCs isolated from umbilical cord blood by immunomagnetic separation were cultured within the porous scaffold in a perfusion bioreactor system. Control cultures were maintained on dishes coated with similar heparin-chitosan films. Oxygen uptake was measured during the culture period. After 7 days of culture, scaffolds were harvested for analysis. Cellular phenotype and HSC characteristics were evaluated via flow cytometry and colony forming unit assays. The results indicate good cell retention and proliferation within the perfused scaffolds. Oxygen consumption in the perfusion bioreactor system increased continuously during the culture, indicating steady cell growth. Cells from the perfused scaffold cultures showed higher percentages of primitive progenitors and exhibited superior colony forming unit performance as compared to cells from static cultures. In addition, perfusion culture at low oxygen (5%) enhanced the expansion of CD34+ cells and colony-forming activity compared to high oxygen (19%) cultures. The results suggest that perfusion culture of cord blood CD34+ cells under bone marrow-like conditions enhances HSC expansion compared to static cultures.

Role of the plasma membrane ROS-generating NADPH oxidase in CD34+ progenitor cells preservation by hypoxia

Hypoxia favored the preservation of progenitor characteristics of hematopoietic stem and progenitor cells (HSPCs) in bone marrow. This work aimed at studying the role of reactive oxygen species (ROS)-generating NADPH oxidase system regulated by hypoxia in ex vivo cultures of cord blood CD34+ cells. The results showed that NADPH oxidase activity and ROS generation were reduced in hypoxia with respect to normal oxygen tension. Meanwhile the ROS generation was found to be inhibited by diphenyleneiodonium (the NADPH oxidase inhibitor), or N-acetylcysteine (the ROS scavenger). Accordingly NADPH oxidase mRNA and p67 protein levels decreased in hypoxia. The analysis of progenitor characteristics, including the proportion of cultured cells expressing the HSPCs marker CD34+CD38-, colony production ability of the colony-forming cells (CFCs), and the re-expansion capability of the cultured CD34+ cells, showed that either 5% pO(2) or reduced ROS favored preserving the characteristics of CD34+ progenitors, and promoted the expansion of CD34+CD38- cells as well. The above results demonstrated that hypoxia effectively maintained biological characteristics of CD34+ cells through keeping lower intracellular ROS levels by regulating NADPH oxidase.

Distinct IL-7 Signaling Outcomes in Normal Human B and T-Lineage Progenitors.

Severe combined immune deficiency (SCID) patients with mutations in the IL-7 receptor (R) alpha chain or the gamma common subunit chain exhibit a severe block in early thymocyte development but have normal numbers of circulating B cells. This phenotype has been interpreted as evidence that human T and B-cell development have different IL-7 signaling requirements. We hypothesize that these apparent differences may reflect activation of different signaling pathways by IL-7, culminating in varying outcomes in survival/proliferation. Thus, the goal of the current study was to test this hypothesis using purified populations of normal human thymic and B-lineage lymphoid progenitors. CD34+ thymocytes (CD1a- multi-lineage lymphoid progenitors and CD1a+ early T-lineage progenitors representing 1–3% of total thymocytes) and CD34− thymocytes (CD4+/CD8+, CD4+/CD8−, and CD4−/CD8+ subpopulations) were purified using anti-CD34 magnetic beads. CD19+ B-lineage cells were purified with anti-CD19 magnetic beads from 4-wk cultures of cord blood CD34+ stem cells plated on MS-5 murine stromal cells. Flow cytometry revealed that the IL-7R was expressed on approx. 95% of CD34+ thymocytes, approx. 50% of CD34− thymocytes, and 30–50% of CD19+ B-lineage cells. Two to 3-day incubation with IL-7 induced proliferation of CD34+ thymocytes, and promoted survival (but not proliferation) of CD34- thymocytes and CD19+ B-lineage cells. Each population was stimulated with log concentrations of IL-7 for 1–20 min, lysed, and Western blotted. CD34+ thymocytes underwent robust phosphorylation (p) of STAT5, ERK1/2, AKT and the AKT substrate GSK3beta within 1 min and peaked at 5–10 min. Signaling was detected with as little as 10 pg/ml IL-7. CD34− thymocytes underwent comparable induction of pSTAT5, but no induction of pERK1/2, pAKT or pGSK3beta at up to 10 ng/ml IL-7. Incubation of CD34+ thymocytes with IL-7 for up to 3 days revealed IL-7 dose-dependent suppression of the cyclin-dependent kinase inhibitor p27Kip1, but no suppression of p27Kip1 was detected in IL-7 stimulated CD34− thymocytes. CD19+ cells exhibited IL-7 dose-dependent induction of pSTAT5, but showed only minimal induction of pERK1/2 and pGSK3beta (and only at 10 ng/ml IL-7), and did not suppress expression of p27Kip1. IL-7 induced pAKT and pERK1/2 in CD34+ thymocytes was blocked by the PI-3 kinase inhibitor LY294002 and the MEK inhibitor U0126, respectively. However, only LY294002 blocked proliferation. IL-7 promoted the expansion of CD19+ B-lineage cells cultured on MS-5 stromal cells, and expansion was blocked by LY294002 but not UO126. These collective results suggest that whereas induction of pSTAT5 is a constant outcome of IL-7 signaling in normal human lymphoid progenitors, activation of PI3-kinase/AKT and subsequent proliferation:occurs in CD34+ thymocytes,does not occur in CD34− thymocytes, andoccurs in CD19+ B-lineage cells; but potentially requires an additional cytokine stimulus derived from MS-5 stromal cells that cooperates with IL-7 to transduce a proliferative signal.These IL-7 signaling outcomes may at least partially explain the differences in T and B lymphocyte development in SCID patients with mutations in the IL-7R alpha chain and the gamma common subunit chain.

MicroRNAs 221 and 222 inhibit normal erythropoiesis and erythroleukemic cell growth via kit receptor down-modulation

MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression primarily through translational repression. In erythropoietic (E) culture of cord blood CD34+ progenitor cells, the level of miR 221 and 222 is gradually and sharply down-modulated. Hypothetically, this decline could promote erythropoiesis by unblocking expression of key functional proteins. Indeed, (i) bioinformatic analysis suggested that miR 221 and 222 target the 3' UTR of kit mRNA; (ii) the luciferase assay confirmed that both miRs directly interact with the kit mRNA target site; and (iii) in E culture undergoing exponential cell growth, miR down-modulation is inversely related to increasing kit protein expression, whereas the kit mRNA level is relatively stable. Functional studies show that treatment of CD34+ progenitors with miR 221 and 222, via oligonucleotide transfection or lentiviral vector infection, causes impaired proliferation and accelerated differentiation of E cells, coupled with down-modulation of kit protein: this phenomenon, observed in E culture releasing endogenous kit ligand, is magnified in E culture supplemented with kit ligand. Furthermore, transplantation experiments in NOD-SCID mice reveal that miR 221 and 222 treatment of CD34+ cells impairs their engraftment capacity and stem cell activity. Finally, miR 221 and 222 gene transfer impairs proliferation of the kit+ TF-1 erythroleukemic cell line. Altogether, our studies indicate that the decline of miR 221 and 222 during exponential E growth unblocks kit protein production at mRNA level, thus leading to expansion of early erythroblasts. Furthermore, the results on kit+ erythroleukemic cells suggest a potential role of these miRs in cancer therapy.

Cord Blood Processing by Using a Standard Manual Technique and Automated Closed System "Sepax" (Kit CS-530)

Stem Cells and DevelopmentVol. 14, No. 1 CorrespondenceCord Blood Processing by Using a Standard Manual Technique and Automated Closed System "Sepax" (Kit CS-530)Bernard Dazey, Pascale Duchez, Catherine Letellier, Gerard Vezon, and Dr. Zoran IvanovicBernard DazeySearch for more papers by this author, Pascale DuchezSearch for more papers by this author, Catherine LetellierSearch for more papers by this author, Gerard VezonSearch for more papers by this author, and Dr. Zoran IvanovicSearch for more papers by this authorPublished Online:22 Feb 2005https://doi.org/10.1089/scd.2005.14.6AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited byDiscarded plasma obtained after cord blood volume reduction as an alternative for fetal calf serum in mesenchymal stromal cells cultures6 August 2020 | Transfusion, Vol. 3Umbilical cord stem cells: Background, processing and applicationsTissue and Cell, Vol. 65Automated thawing increases recovery of colony-forming units from banked cord blood unit grafts11 October 2018 | Transfusion, Vol. 58, No. 12Cord Blood Graft Thawing14 October 2017Comparison of human umbilical cord blood processing with or without hydroxyethyl starch23 August 2017 | Transfusion, Vol. 57, No. 11Cord blood collection and processing with hydroxyethyl starch or non–hydroxyethyl starchCytotherapy, Vol. 18, No. 5Cell BankingCurrent thawing and infusion practice of cryopreserved cord blood: the impact on graft quality, recipient safety, and transplantation outcomes4 June 2014 | Transfusion, Vol. 54, No. 11A Novel Procedure to Improve Functional Preservation of Hematopoietic Stem and Progenitor Cells in Cord Blood Stored at +4°C Before Cryopreservation Jean Chevaleyre, Laura Rodriguez, Pascale Duchez, Marie Plainfossé, Bernard Dazey, Véronique Lapostolle, Marija Vlaski, Philippe Brunet de la Grange, Bruno Delorme, and Zoran Ivanovic7 April 2014 | Stem Cells and Development, Vol. 23, No. 15Cord blood banking activity in Iran National Cord Blood Bank: A two years experienceTransfusion and Apheresis Science, Vol. 50, No. 1Good manufacturing practice-compliant isolation and culture of human umbilical cord blood-derived mesenchymal stem cellsJournal of Translational Medicine, Vol. 12, No. 1Nutrient Regulation by Continuous Feeding Removes Limitations on Cell Yield in the Large-Scale Expansion of Mammalian Cell Spheroids18 October 2013 | PLoS ONE, Vol. 8, No. 10Cryopreservation of hematopoietic stem and progenitor cells amplified ex vivo from cord blood CD34+ cells11 December 2012 | Transfusion, Vol. 53, No. 9Functional Stability (at +4°C) of Hematopoietic Stem and Progenitor Cells Amplified Ex Vivo from Cord Blood CD34 + Cells1 August 2013 | Cell Transplantation, Vol. 22, No. 8Potential Application of Cord Blood-Derived Stromal Cells in Cellular Therapy and Regenerative MedicineJournal of Blood Transfusion, Vol. 2012Definitive Setup of Clinical Scale Procedure for Ex Vivo Expansion of Cord Blood Hematopoietic Cells for Transplantation1 November 2012 | Cell Transplantation, Vol. 21, No. 11Human Umbilical Cord Blood-Derived Very-Small-Embryonic-Like Stem Cells with Maximum Regenerative Potential? 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In Vitro Culture during Retroviral Transduction Improves Thymic Repopulation and Output after TBI and Autologous PBPC Transplantation in Rhesus Macaques.

We recently completed a longitudinal study over 12 months on T-cell immune reconstitution after total body irradiation and autologous transplantation of peripheral blood progenitor cells in 3–5 year old rhesus macaques. The focus of the study was to evaluate the source of T-cell recovery using three different sources of cells for transplantation. The starting number of CD34+ cells contained within the graft was adjusted to be the same for each of the three groups: unselected peripheral blood progenitor cells (PBPC) (n=3) vs. CD34+ selected mobilized PBPC (n=4) vs. CD34+ selected cells cultured for 4 days in vitro (Flt-3L/MDGF/SCF/retronectin) and retrovirally-transduced (n=3). Peripheral blood and lymph nodes were collected for phenotypic and TREC analysis. There was a trend for animals receiving cultured and transduced CD34+ cells to attain higher absolute naive and memory CD4+ T-cell, CD20+ B-cell and CD16+ NK-cell numbers in the first months after transplantation compared to the two other groups. There were no differences for numbers of CD8+ T-cells, monocytes or dendritic cells. The absolute TREC amount per μl blood of the CD4+ T-cells in the group that received selected-transduced cells was significantly higher than in the group that received unselected cells (p=0.0166) and for CD8+ cells significantly higher than for selected cells (p=0.0464). There was significantly less peripheral T-cell expansion as measured by Ki-67 expression in the selected-transduced group compared to the two other groups: At 1 month after transplantation the mean Ki-67 expression level for CD8+ cells in peripheral blood was 33.9% in the selected vs. 23.3% in the unselected vs. 8% in the selected-transduced group. Histology at 12 months revealed striking differences in the thymus between the 3 groups, while other organs (lymph nodes, spleen, tonsils and Peyer's patches) showed no remarkable differences. In the CD34+ selected-transduced group the thymus showed preserved lobular architecture with well defined cortical and medullary areas, compared to atrophy with fat replacement, decreased thickness of the cortex, and cystic changes of the thymic epithelium in the CD34+ selected and unselected groups. The degree of atrophy was more pronounced in the latter group. This study demonstrates an enhanced ability of in vitro expanded and retrovirally transduced cells to repopulate the thymus compared to non-manipulated CD34+ selected cells. In vitro expansion and transduction may promote development of T-cell and NK-cells as has been described earlier for fetal thymic organ culture of cord blood CD34+ cells as well as in the SCID-hu mouse model. Our observation of an acceleration of T-cell and NK cell immune reconstitution following in vitro culture contrasts with limited prior clinical trial experiences describing poor repopulating potential of cultured progenitor cells. Our data suggest that in vitro culture and retroviral gene transfer per se does not induce an intrinsic differentation defect. Future studies are planned to examine mechanisms behind the improved repopulation ability of selected-transduced progenitor cells, which has important implications for gene therapy trials, and suggests that cultured cells may be useful for a number of clinical applications.

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.

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.

CD13/N-aminopeptidase is involved in the development of dendritic cells and macrophages from cord blood CD34+ cells

Expression of CD13/N-aminopeptidase may reflect cell activation and growth. We examined its role regarding cell growth in cultures of cord blood CD34(+) cells with stem cell factor/Flt-3 ligand/granulocyte-macrophage colony-stimulating factor/tumor necrosis factor-alpha. Indeed, 82% +/- 6% of cells from culture day 5 were CD13(hi), 25% +/- 8% of which were still Lin-. About 50% of CD13(hi)Lin- cells, which comprise progenitors of dendritic cells (DC), monocytes/macrophages and granulocytes, and 30% of CD13(lo)Lin- cells were CD34(+). Sorted CD34(+)CD13(hi)Lin- cells, cultured further for 7 days with the same cytokines, expanded 31-fold and CD34(-)CD13(hi)Lin- cells 7-fold, but CD34(+)CD13(lo)Lin- and CD34(-)CD13(lo)Lin- cells did not grow. Thus, cell growth correlated with CD13 expression, all the more so that cells were CD34(+). Actinonin, the most potent N-aminopeptidase inhibitor, was used to engage CD13 on sorted CD13(hi)Lin- cells and on culture day-7 bulk cells. In both cases, this resulted in reversible cell growth arrest, with 30% to 60% fewer cells in the G2/S-M phase than in controls. Interestingly, similar effects were noted with CD13 monoclonal antibody TUK1, which does not inhibit N-aminopeptidase activity, but not with N-aminopeptidase-blocking antibodies WM15 and F23. All cycling cells appeared susceptible to actinonin, which induced cell apoptosis at the same time as Bcl-2 was downregulated and caspase-3 activity increased, but finally percentages and yields of DC and macrophage precursors were affected more than those of granulocytic cells. Thus, through engagement of N-aminopeptidase enzymatic site but possibly also of an independent determinant, CD13 plays a role in the growth of DC/macrophage progenitors and precursors. (Blood. 2000;95:453-460)

Is in vitro expansion of human cord blood cells clinically relevant?

Peripheral blood recovery after cord blood (CB) transplantation is delayed compared with marrow. Expansion of CB haemopoietic cells has been investigated with the aim of reducing cytopenia following transplantation. Mature cells, post-progenitors, progenitors and long-term culture-initiating cells (LTC-IC) from expansion products may contribute to peripheral blood recovery. We investigated the increase in total nucleated cells, colony-forming cells (CFC), CD34+ cells and LTC-IC by limiting dilution after a 14 day culture of CB CD34+ cells (5 x 10(3)/ml) with SCF, IL-3, IL-6, GM-CSF and G-CSF all at 10 ng/ml. On average, nucleated cells increased 2500-fold, CD34+ cells 39-fold and CFU-GM 49-fold with maintenance of BFU-E. The more primitive LTC-IC expanded on average 2.5-fold but effects on long-term marrow-repopulating cells (LTRC) during culture are unknown. A practical application of in vitro expansion of CB might be to expand a 20% aliquot of a CB donation and infuse the remainder unmanipulated. This could provide a 5- to 7-fold increase in progenitor cells, an estimated 1570-fold increase in post-progenitor cells and maintenance of LTC-IC compared to an untreated donation. Combined with in vivo post-transplant growth factor therapy this could prompt early peripheral blood recovery after CB transplantation, without significant loss of LTC-IC or donor lymphocytes.

Synergistic Action of Flt3 and gp130 Signalings in Human Hematopoiesis

AbstractWe recently showed that c-kit signal synergizes with glycoprotein (gp)130 signal mediated by a complex of interleukin (IL)-6 and soluble IL-6 receptor (IL-6/sIL-6R) to stimulate the expansion of human primitive hematopoietic progenitor cells and erythropoietin-independent erythropoiesis. In the present study, we examined the effect of a ligand for Flt3 (FL), whose receptor tyrosine kinase is closely related to c-kit, in combination with IL-6/sIL-6R on human hematopoiesis in vitro. In serum-containing methylcellulose clonal culture of cord blood CD34+ cells, whereas FL alone stimulated only granulocyte-macrophage (GM) colony formation, erythroid bursts and mixed colonies in addition to GM colonies were induced by FL with IL-6/sIL-6R, but not IL-6/sIL-6R alone. In suspension culture, CD34+ cells generated a small number of myeloid cells in the presence of FL or IL-6/sIL-6R alone. However, the addition of IL-6/sIL-6R to the culture with FL induced the generation of a significant number of erythroid cells and megakaryocytes in addition to myeloid cells. The combination of FL and IL-6/sIL-6R also induced a remarkable expansion of GM colony- and erythroid burst-forming cells and multipotential progenitors, although FL or IL-6/sIL-6R alone induced the generation of only a small number of progenitors for GM colonies. The synergistic effects of FL and IL-6/sIL-6R were confirmed in serum-free clonal and suspension cultures. In addition, the addition of anti-human gp130 monoclonal antibodies abrogated the synergistic action. These results indicate that Flt3 signal, as well as c-kit signal, synergizes with gp130 signal to stimulate human myelopoiesis, erythropoiesis and megakaryopoiesis, and the expansion of primitive multipotential hematopoietic progenitor cells.

Synergistic Action of Flt3 and gp130 Signalings in Human Hematopoiesis

We recently showed that c-kit signal synergizes with glycoprotein (gp)130 signal mediated by a complex of interleukin (IL)-6 and soluble IL-6 receptor (IL-6/sIL-6R) to stimulate the expansion of human primitive hematopoietic progenitor cells and erythropoietin-independent erythropoiesis. In the present study, we examined the effect of a ligand for Flt3 (FL), whose receptor tyrosine kinase is closely related to c-kit, in combination with IL-6/sIL-6R on human hematopoiesis in vitro. In serum-containing methylcellulose clonal culture of cord blood CD34+ cells, whereas FL alone stimulated only granulocyte-macrophage (GM) colony formation, erythroid bursts and mixed colonies in addition to GM colonies were induced by FL with IL-6/sIL-6R, but not IL-6/sIL-6R alone. In suspension culture, CD34+ cells generated a small number of myeloid cells in the presence of FL or IL-6/sIL-6R alone. However, the addition of IL-6/sIL-6R to the culture with FL induced the generation of a significant number of erythroid cells and megakaryocytes in addition to myeloid cells. The combination of FL and IL-6/sIL-6R also induced a remarkable expansion of GM colony- and erythroid burst-forming cells and multipotential progenitors, although FL or IL-6/sIL-6R alone induced the generation of only a small number of progenitors for GM colonies. The synergistic effects of FL and IL-6/sIL-6R were confirmed in serum-free clonal and suspension cultures. In addition, the addition of anti-human gp130 monoclonal antibodies abrogated the synergistic action. These results indicate that Flt3 signal, as well as c-kit signal, synergizes with gp130 signal to stimulate human myelopoiesis, erythropoiesis and megakaryopoiesis, and the expansion of primitive multipotential hematopoietic progenitor cells.

Interleukin-3 Cooperates With Tumor Necrosis Factor α for the Development of Human Dendritic/Langerhans Cells From Cord Blood CD34+ Hematopoietic Progenitor Cells

We have previously shown that tumor necrosis factor (TNF)alpha strongly potentiates the granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin (IL)-3-dependent proliferation of CD34+ hematopoietic progenitor cells (HPC) through the recruitment of early progenitors with high proliferative potential. Furthermore, the combination of GM-CSF and TNFalpha allows the generation of large numbers of dendritic/Langerhans cells (D-Lc). Herein, we analyzed whether IL-3, when combined to TNFalpha would, as does GM-CSF, allow the generation of CD1a+ D-Lc. Accordingly, cultures of cord blood CD34+ HPC with IL-3 + TNFalpha yielded 20% to 60% CD14+ cells and 11% to 17% CD1a+ cells, while IL-3 alone did not generate significant numbers of CD1a+ cells. Although the percentage of CD1a+ cells detected in IL3 + TNFalpha was lower than that observed in GM-CSF + TNFalpha (42% to 78%), the strong growth induced by IL-3 + TNFalpha generated as many CD1a+ cells as did GM-CSF + TNFalpha. The CD14+ and CD1a+ cells generated with IL-3 + TNFalpha are similar to CD14+ and CD1a+ cells generated in GM-CSF alone and GM-CSF + TNFalpha, respectively. CD1a+ cells differed from CD14+ cells by (1) dendritic morphology, (2) higher expression of CD1a, CD1c, CD4, CD40, adhesion molecules (CD11c, CD54, CD58), major histocompatibility complex (MHC) class II molecules and CD28 ligands (CD80 and CD86), (3) lack of Fc receptor FcgammaRI (CD64) and complement receptor CR1 (CD35) expression, and (4) stronger induction of allogeneic T-cell proliferation. Thus, in combination with TNFalpha, IL-3 is as potent as GM-CSF for the generation of CD1a+ D-Lc from cord blood CD34+ HPC. The dendritic cell inducing ability of IL-3 may explain why mice with inactivated GM-CSF gene display dendritic cells.