Umbilical Cord Blood Hematopoietic Progenitor Research Articles

436 - Ex Vivo Epigenetic Modification with Valproic Acid (VPA) Plus Nicotinamide (NAM) Results in Expansion of Umbilical Cord Blood Hematopoietic Progenitors Leading to Faster Engraftment and Chimerism in In Vivo Mouse Xenograft Model

436 - Ex Vivo Epigenetic Modification with Valproic Acid (VPA) Plus Nicotinamide (NAM) Results in Expansion of Umbilical Cord Blood Hematopoietic Progenitors Leading to Faster Engraftment and Chimerism in In Vivo Mouse Xenograft Model

Over-Expression of the Local Bone Marrow Renin-Angiotensin System in Acute Myeloid Leukemia.

Local bone marrow (BM) renin-angiotensin system (RAS) is an autocrine-paracrine system affecting hematopoiesis [Haznedaroglu IC, et al. A local RAS in the bone marrow. MedHypotheses. 1996; 46:507–10., Haznedaroglu IC. A local RAS in the bone marrow still awaits its Christopher Columbus. ExpHematol. 1998; 26:279., Haznedaroglu IC, et al.Towards the understanding of the local hematopoietic bone marrow RAS. IntJBiochemCellBiol. 2003; 35:867–80.] Angiotensin II type 1a (AT1a) receptors are present on the CD34+ hematopoietic stem cells from the BM or cord blood (CB) [Goker H, et al. Local umbilical cord blood renin-angiotensin system. AnnHematol . 2005; 84:277–81]. Angiotensin II stimulates the proliferation of BM and umbilical CB hematopoietic progenitors. Local RAS may also be involved in leukemogenesis and polycythemia vera [Aksu S, et. al. Enhanced Expression of the Local Haematopoietic Bone Marrow Renin-Angiotensin System in Polycythemia Rubra Vera J Int Med Res (in press), Aksu S, et. al. Over-expression of angiotensin converting enzyme (CD143) on leukemic blasts as a clue for the activated local bone marrow RAS in AML. Leukemia and Lymphoma (in press)]. Angiotensin converting enzyme (ACE) hyperfunction may lead to the acceleration of negative hematopoietic regulator peptide, AcSDKP, metabolism, which in turn lowers its level in the bone marrow microenvironment, finally removing the anti-proliferative effect of AcSDKP on the hematopoietic cells and blasts. Renin expression could have a role in leukemia development and angiotensin may act as an autocrine growth factor for AML cells. In this study, the expression of the mRNAs of the major RAS components, namely ACE, renin, and angiotensinogen in human BM samples were quantified by RT-PCR to confirm the presence of the local BM RAS. Furthermore, we assessed quantitative alterations of the ACE, renin, and angiotensinogen mRNAs in leukemic BM samples. The aim of this study was to search those major RAS components in the leukemic blast cells taken from the BM of the patients with AML. BM aspirations were obtained from 10 patients with AML and 8 patients with non-malignant hematological disorders. All of three gene expressions were found to be significantly higher in the BM samples of AML patients compared to the BM samples of the control group (Figure 1). Pathological activity of the local RAS-mediated regulation may be important, since the angiotensin peptides represent a molecular target in the disease management. [Display omitted]

Effect of Blood Components and Obstetric Variables on the Viability and Clonogenicity of Umbilical Cord Blood Hematopoietic Progenitors

Effect of Blood Components and Obstetric Variables on the Viability and Clonogenicity of Umbilical Cord Blood Hematopoietic Progenitors

The effects of Gremlin1 on human umbilical cord blood hematopoietic progenitors.

Bone morphogenetic proteins (BMPs) support malignant hematopoiesis in CML. Conversely, the multi-functional BMP antagonist Gremlin1 supports self-renewing cancer stem cells of other malignancies. Inhibition of BMP signaling in CML, or of Gremlin1 in solid tumors, may therefore have therapeutic potential. However, since BMPs regulate hematopoietic stem cell (HSC) decisions in the stem cell niche, it is necessary to determine how Gremlin1 influences normal HSC. We examined the effects of Gremlin1 on long-term culture-initiating cells (LTC-IC) and transplantable hematopoietic stem cells (SCID-repopulating cells: SRC) in human umbilical cord blood. Gremlin1 inhibited BMP signaling, downregulated BMP-6 and cyclin E2 expression and upregulated hairy and enhancer of split-1 (HES-1; a Notch transcriptional target) and Hedgehog interacting protein-1 (HHIP-1; an inhibitor of Hedgehog signaling). The functional effects of Gremlin1 on SRC, i.e. skewing of their myelopoietic:lymphopoietic potential towards B lymphopoiesis without affecting long-term engraftment potential, were entirely consistent with changes in gene expression induced by Gremlin1. Since both BMPs and Gremlin1 are secreted by osteoblasts in vivo, our studies provide potential insights into the molecular regulation of hematopoiesis in the stem cell niche. These results also suggest that Gremlin1 (and possibly its mimetics that may be developed for therapeutic use) may not adversely affect normal human hematopoietic stem cell survival, though they may reduce their myelopoietic potential.

050 Meeting medical device standards with a closed system cryogenic Vial for Cellular Therapy Products

The use of closed systems for low volume processing and storage of cell therapy products is necessary for good manufacturing practices (GMP), yet compliance with medical device regulatory test standards is lacking. A sterile closed system vial (CellSeal® cryogenic vial, Cook General BioTechnology, LLC, IN, USA) that meets pharmaceutical quality requirements was evaluated to determine if the vial also meets medical device standards for storage of cellular therapy products at cryogenic temperatures. The post-sterile stability and shelf-life of the device was evaluated by subjecting the device packaging to hazardous situations (manufacturing, transportation, distribution, and aging) typically undergone prior to usage. The packaging was exposed to simulated dynamic environmental conditioning, and separately to aging conditions equal to 36 real-time months. Subsequent to both dynamic environmental conditioning and aging, a series of product tests were completed to determine if the device maintained sterility and strength/integrity throughout the hazardous environments. Also, the device was evaluated to address necessary related and potential biological and toxicological hazards associated with the device intended use. Additionally, the loaded, sealed, and frozen device was tested for durability and integrity utilizing a 1-meter drop test. In addition, these were transported and tested using pharmaceutical packaging tests including dye ingress and microbial challenge. Further, human umbilical cord blood hematopoietic progenitor cells (UCB) from 3 donors were used to establish the performance of the vials after 1-month of storage. For each donor, a 30 ml concentrated UCB was prepared after adding 10% dimethyl sulfoxide as cryoprotectant. Next, 10 ml of this was cryopreserved using 2 × 5ml vials with the remaining 20 ml cryopreserved in standard FDA-cleared Pall® Medical cord blood freezing bags as a control. For both CellSeal vials and bags, an integral test segment was retained. All samples were analyzed for total nucleated cell count, colony-forming unit (CFU) assay and viability using 7-AAD. This study confirms that the CellSeal cryogenic vial meets medical device testing standards for low volume cryopreservation of clinical cell therapy products. Source of funding: Cook General BioTechnology, LLC, 1102 Indiana Avenue, Indianapolis, IN, USA Conflict of interest: None declared. evonne.fearnot@cookgbt.com

Evaluation of potential ionizing irradiation protectors and mitigators using clonogenic survival of human umbilical cord blood hematopoietic progenitor cells

We evaluated the use of colony formation (colony-forming unit-granulocyte macrophage [CFU-GM], burst-forming unit erythroid [BFU-E], and colony-forming unit-granulocyte-erythroid-megakaryocyte-monocytes [CFU-GEMM]) by human umbilical cord blood (CB) hematopoietic progenitor cells for testing novel small molecule ionizing irradiation protectors and mitigators. The following compounds were added before (protection) or after (mitigation) ionizing irradiation: GS-nitroxides (JP4-039 and XJB-5-131), the bifunctional sulfoxide MMS-350, the phosphoinositol-3-kinase inhibitor LY29400, triphenylphosphonium-imidazole fatty acid, the nitric oxide synthase inhibitor (MCF-201-89), the p53/mdm2/mdm4 inhibitor (BEB55), methoxamine, isoproterenol, propranolol, and the adenosine triphosphate-sensitive potassium channel blocker (glyburide). The drugs XJB-5-131, JP4-039, and MMS-350 were radiation protectors for CFU-GM. JP4-039 was also a radiation protector for CFU-GEMM. The drugs XJB-5-131, JP4-039, and MMS-350 were radiation mitigators for BFU-E, MMS-350 and JP4-039 were mitigators for CFU-GM, and MMS350 was a mitigator for CFU-GEMM. In contrast, other drugs were effective in murine assays; TTP-IOA, LY294002, MCF201-89, BEB55, propranolol, isoproterenol, methoxamine, and glyburide but showed no significant protection or mitigation in human CB assays. These data support the testing of new candidate clinical radiation protectors and mitigators using human CB clonogenic assays early in the drug discovery process, thus reducing the need for animal experiments.

NT-3 and IGFBP-2 increase the expansion of umbilical cord blood hematopoietic progenitors

NT-3 and IGFBP-2 increase the expansion of umbilical cord blood hematopoietic progenitors

Time and temperature before processing influence the recovery of umbilical cord blood hematopoietic progenitors

Time and temperature before processing influence the recovery of umbilical cord blood hematopoietic progenitors

Over-expression of angiotensin-converting enzyme (CD 143) on leukemic blasts as a clue for the activated local bone marrow RAS in AML

Local bone marrow renin-angiotensin system (RAS) is an autocrine-paracrine system affecting hematopoiesis. Angiotensin II type 1a (AT1a) receptors are present on the CD34+ hematopoietic stem cells. Angiotensin II stimulates the proliferation of bone marrow and umbilical cord blood hematopoietic progenitors. There are preliminary data that local RAS might also be involved in leukemogenesis. ACE hyper-function may lead to the acceleration of negative hematopoietic regulator peptide, AcSDKP, metabolism, which in turn lowers its level in the bone marrow micro-environment, finally removing the anti-proliferative effect of AcSDKP on the hematopoietic cells and blasts. Renin expression could have a role on the leukemia development and angiotensin may act as an autocrine growth factor for acute myeloid leukemia (AML) cells. The aim of this study is to search ACE (CD 143) surface antigen by flow-cytometric analyses on the leukemic blast cells taken from the bone marrow of the patients with AML. Bone marrow aspiration materials and peripheral blood samples were obtained from 11 patients with AML (eight males, three females; aged 46 (range 26–67) years) and six patients with non-malignant hematological disorders (four males, two females; aged 56 (range 22–71) years). ACE (CD 143) surface antigen was shown to be over-expressed in leukemic myeloid blast cells. ACE is positively correlated with bone marrow blast count. Elucidation of the pathological activity of the local RAS-mediated regulation of the leukemogenesis is both pathobiologically and clinically important, since the angiotensin peptides represent a molecular target in the disease management.

Cocultures of Umbilical Cord Blood Hematopoietic Progenitor Cells and Umbilical Cord Endothelium.

Purpose. The expansion of hematopoietic progenitor cells (HPC) from cord blood is limited so far. For HPC expansion and differentiation we evaluated a coculture model of cord blood-derived HPC and endothelial cells (EC) isolated from the umbilical cord.Methods. CD34(+) HPC were cultured directly in contact with the endothelial monolayer or indirectly using a 0.4μm microporous transmembrane. EC were stimulated by various cytokines including TPO, interleukin (IL) 1β, IL4, and IL6. After 1 and 2 weeks, the HPC were counted and analyzed by flow cytometry. Their plating efficiencies were determined by methylcellulose assays, and their reconstitutive potential was analyzed by cobblestone assays. DNA microarrays on the expanding cells as well as on cytokine-stimulated EC were also performed.Results. EC stimulation with thrombopoietin and IL4 had no significant effect on mononuclear cell expansion. In contrast, IL1β stimulation resulted in a 10 and 100fold increase of the cell number after one and two weeks, respectively. More than 90% of these cells were CD33(+) and had a plating efficiency equivalent to that of HPC post isolation (7.3 ±1.2 versus 8.3±0.68%). The number of 5-wk cobblestone-area forming cells among these expanded cells was diminished, whereas the number of 2-wk cobblestone areas did not change (10.6 versus 8.5). The expanded cells could further mature into functional granulocytes. Time-course DNA microarrays of IL1β-stimulated EC demonstrated an increased expression of genes encoding tumor necrosis factor-induced proteins, interleukins, and endothelial adhesion molecules. When IL1β was replaced by IL6, a 7-fold expansion of morphologically progenitor-like cells took place. 10–13% of these cells were positive for CD133, had a plating efficiency of 10.2±1.7%, and showed on average 14.5 five-week cobblestone areas.Conclusion. Cocultures of cord blood-derived HPC with umbilical cord-derived EC offer alternative methods for the expansion and differentiation of hematopoietic cells.

Characterization of banked umbilical cord blood hematopoietic progenitor cells and lymphocyte subsets and correlation with ethnicity, birth weight, sex, and type of delivery: a Cord Blood Transplantation (COBLT) Study report

The Cord Blood Transplantation (COBLT) Study banking program was initiated in 1996. The study goals were to develop standard operating procedures for cord blood (CB) donor recruitment and banking and to build an ethnically diverse unrelated CB bank to support a transplantation protocol. The hematopoietic progenitor cell (HPC) and lymphocyte subset (LS) content of approximately 8000 CB units were characterized, and these results were correlated with donor ethnicity, birth weight, gestational age, sex, and type of delivery. There was a significant correlation of CD34+ cell count with colony-forming unit (CFU)-granulocyte-macrophage (r=0.68, p<0.001), CFU-granulocyte-erythroid-macrophage-megakaryocyte (r=0.52, p<0.001), burst-forming unit-erythroid (BFU-E; r=0.61, p<0.001), and total CFUs (r=0.67, p<0.001). Nucleated red blood cell count was significantly correlated with total CD34+ (r=0.56, p<0.001), total CFU (r=0.50, p<0.001), BFU-E (r=0.48, p<0.001), and counts of CD34+ subsets (p<0.001). Caucasian ethnicity was significantly correlated with higher CD3+/CD4+, CD19+, and CD16+/CD56+ LSs. Furthermore, CD34+/CD38- and CD34+/CD61+ CB units (HPC-C) were significantly lower in African American and Asian persons compared to Caucasian and Hispanic persons. Male sex was associated with significantly fewer CD3+/CD4+, CD19+, and CD16+/CD56+ but increased CD3+/CD8+ LSs (p<0.001). Finally, cesarean section was associated with significantly higher total CFU and CD16+/CD56+ but lower CD3+/CD4+, CD3+/CD8+, and CD19+ LSs. These results provide a standard and range for uniformly processed HPC-C progenitor cells and LSs. CB progenitor cells and/or LSs may in the future predict for rapidity of engraftment, incidence of graft-versus-host disease, speed and quality of immunore- constitution, graft-versus-tumor effects, and/or success of gene transfection after CB transplantation.

A theoretically optimized method for cord blood stem cell cryopreservation.

The objective of this study was to develop an optimal cryopreservation method for human umbilical cord blood hematopoietic progenitor cells as evidenced by improved retention of in vivo engraftment ability and multilineage differentiation. An extended understanding of the osmometric/permeability characteristics of cord blood stem cells was accomplished by measuring permeability of the cryoprotectant dimethyl sulfoxide (DMSO) at below-ambient temperatures (10 degrees and 3 degrees C). These data were combined with previously published osmotic and permeability data and the water-NaCl-DMSO phase diagram in conjunction with a mathematical model to determine an optimal initial DMSO concentration, cooling rate, and liquid nitrogen plunging temperature. Cells cryopreserved with the theoretically optimized procedure were then compared with cells frozen using standard methods for the ability to engraft in irradiated NOD/SCID mice. The optimal procedure was determined to include a 0.7 molal (approximately 5%) DMSO concentration at a cooling rate of 4 degrees C/min, and a plunging temperature of -44 degrees C. The optimized protocol resulted in significantly higher engraftment of human CD45(+) cells (17.2 +/- 1.6% vs. 8.4 +/- 1.6%), CD19(+) B lymphocytes (11.3 +/- 1.2% vs. 5.8 +/- 1.2%), and CD34(+) cells (1.9 +/- 0.09% vs. 0.6 +/- 0.09%) compared to cells frozen using a standard method. Engraftment of CD33(+) cells was not significantly different (4.0 +/- 0.3 vs. 3.2 +/- 0.6, respectively). This study demonstrated that the use of a theoretically determined optimal cryopreservation method is superior to standard methods for maintaining UCB PCBs with multilineage repopulation potential in NOD/SCID mice.

Expression of type 1 (interferon gamma) and type 2 (interleukin-13, interleukin-5) cytokines at distinct stages of natural killer cell differentiation from progenitor cells

To determine whether production of type 1 and type 2 cytokines defines discrete stages of natural killer (NK) cell differentiation, cytokine expression was analyzed in human NK cells generated in vitro in the presence of interleukin-15 (IL-15) and/or IL-2 from umbilical cord blood hematopoietic progenitors. Like peripheral NK cells, the CD161(+)/CD56(+) NK cells from these cultures contained a tumor necrosis factor alpha (TNF-alpha)(+)/granulocyte macrophage-colony-stimulating factor (GM-CSF)(+) subset, an interferon gamma (IFN-gamma)(+) subset, mostly included within the former, and very few IFN-gamma(-)/IL-13(+) cells. Instead, most immature CD161(+)/CD56(-) NK cells, detectable only in the cultures with IL-2, produced IL-13, TNF-alpha, and GM-CSF, but not IFN-gamma, and contained an IL-5(+) subset. In short-term cultures with IL-12 and feeder cells, a proportion of the immature cells acquired the ability to produce IFN-gamma. Part of these produced both IFN-gamma and IL-13, irrespective of induced CD56 expression. These in vitro data indicate that ability to produce the type 2 cytokines IL-13 and IL-5 defines CD161(+) NK cells at intermediate stages of differentiation, and is lost upon terminal functional differentiation, concomitant with acquired ability to produce IFN-gamma.

Ex vivo expansion of human umbilical cord blood hematopoietic progenitor cells in a novel three-dimensional culture system

A novel three-dimensional culture system for the ex vivo expansion of human umbilical cord blood (CB) hematopietic progenitor cells (HPCs) was developed by growing CB mononuclear cells on highly porous CultiSpher G microspheres coated with human bone marrow stromal cells in stirred flasks in the presence of supplemented cytokines. After 12 days, the number of total viable cells, colony-forming units in culture (CFU-C) and CD34+ cells present in the cultures reflected average increases of 7.7, 23.3 and 9.6-fold, respectively, and marked hematopoietic islands were formed on the surface of CultiSpher G.

In vitro study on the cloning and transduction of human O6-methylguanine-DNA-methyltransferase cDNA into human umbilical cord blood CD34+ cells

Objective: To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human O6-methylguanine-DNA-methyltransferase (MGMT) gene could increase resistance to 1,3-Bis(2- Chloroethyl)-l-Nitrosourea (BCNU). Methods: The eDNA encoding the MGMT was isolated by using RTPCR method from total RNA of fresh human liver, the fragment was cloned into pGEM-T vector and further subcloned into GINa retrovirus vector. Then the G1Na- MGMT was transduced into the packaging cell lines GP+E86 and PA317 by LipofectAMINE. By using the medium containing BCNU for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone, high titer amphotropic PA317 producer clone with the highest titer up to 5.8×105 CFU/ml was obtained. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human MGMTcDNA under stimulation of hemopoietic growth factors. Results: The retrovirus vector construction was verified by restriction endonuclease analysis and DNA sequencing. PCR, RT-PCR, Southern Blot, Western Blot and MTT analyses showed that MGMT drug resistance gene has been integrated into the genomic DNA of cord blood CD34 + cells and expressed efficiently. The transgene cord blood CD34 + cells conferred 4-folds stronger resistance to BCNU than untransduced cells. Conclusion: The retrovirus vector-mediated transfer of MGMT drug resistance gene into human cord blood CD34+ cells and its expression provided an experimental foundation for gene therapy in clinical trial.

Delineation of human cord blood hematopoietic progenitor cell subsets with a unique monoclonal antibody AY19.

AY19, a unique mAb was used to better characterize the umbilical cord blood hematopoietic progenitor subpopulations. This mAb identifies an 85 kDa cell surface glycoprotein. In this present study we showed that AY19 mAb is reactive with 50-60% of the CD34+ cord blood cells. Extensive phenotypical studies revealed that AY19 mAb defines a novel CD34+ subset different from the ones defined by anti-CD90, anti-CD38, anti-CD33, anti-CD71, anti-CD19, anti-CD7 or anti-HLA-DR mAbs. We show that AY19 mAb reacts with both primitive and committed progenitors including myeloid and lymphoid progenitors. In addition, sorted CD34+high/AY19+ cells contain an increased number of CFU-GM and a decreased number of BFU-E compared with sorted CD34+high/AY19- cells. We show that AY19 mAb exhibits agonistic properties by inducing a significant increase in the size of CFU-GM colonies when added to serum-free liquid cultures of hematopoietic progenitors. This suggests that AY19 mAb identifies a cell surface receptor which may be involved in the regulation of hematopoietic cell proliferation.

Ex vivo expansion of umbilical cord blood hematopoietic progenitor cells by combinations of cytokines

Ex vivo expansion of hematopoietic progenitor cells in the umbilical cord blood mononuclear cells (CB-MNC) was investigated in liquid culture system with various combinations of cytokines (stem cell factor [SCF], interleukin [IL]-3, IL-6, granulocyte-colony stimulating factor [G-CSF], erythropoietin [EPO], and interferon [INF]-gamma). Non-lineage-committed hematopoietic progenitor cells and lineage committed hematopoietic progenitor cells were represented as CD34+CD38- and CD34+CD38+ subpopulations, respectively. Although absolute CD34+CD38- cell numbers decreased even in the presence of multicytokines, the combinations of SCF plus IL-6 and SCF plus IL-3 plus IL-6 plus IFN-gamma were significantly effective in maintaining CD34+CD38- cells than the other combinations (P < 0.05). After 4 weeks of culture. CD34+CD38- cells disappeared in all combinations of cytokines. Absolute CD34+CD38+ cell numbers increased in the presence of cytokines. Maximal expansion of CD34+CD38+ cells were observed in the combinations of SCF plus IL-3 plus IL-6 plus EPO (19.8 +/- 3.3-fold) and SCF plus IL-3 plus IL-6 plus G-CSF (18.3 +/- 2.6). The combination of SCF plus IL-3 plus IL-6 was also effective to expand CD34+CD38- cells (15.8 +/- 3.9). However, the expansion was transient and they decreased to zero within 3 weeks. In the combinations of SCF plus IL-6 and SCF plus IL-3 plus IL-6 plus INF-gamma, maximal expansion was inferior to the others but CD34+CD38+ cells were maintained more than 4 weeks. These results suggested that the indication of CBT can be expanded into older children by ex vivo augmentation of CB hematopoietic progenitor cells using multi-cytokines.

Enrichment, Characterization, and Responsiveness of Single Primitive CD34+++ Human Umbilical Cord Blood Hematopoietic Progenitors With High Proliferative and Replating Potential

To characterize the growth of cord blood progenitor cells, single nonadherent, low-density, T-lymphocyte-depleted CD34 cells were sorted by flow cytometer with an autoclone device into single wells containing culture medium and cytokines. These cells were evaluated for proliferation and for replating ability of their progeny. This latter effect is used as a measure of self-renewal capacity. Colony formation was assessed in 1 degree wells containing various cytokines, alone and in combination, and single colonies deriving after 21 days in semisolid medium were replated into 2 degree wells in the presence of the combination of purified preparations of recombinant human steel factor (SF, a c-kit ligand), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3), and erythropoietin (Epo). Replating of single colonies was performed also for 3 degrees, 4 degrees, and 5 degrees cultures. In the presence of serum, colony formation was observed in > 66% of the wells stimulated with the combination of Epo, SF, GM-CSF, G-CSF, and IL-3, and more than 39% of the colonies formed in these 1 degree wells were very large in size (> 2.5 mm in diameter, dense in the center, and containing > 10(4) cells/colony). The replating efficiency of these large colonies was up to 93% with generation of subsequent colonies of very large size. Replating could be shown for up to five generations. The cells in these colonies were large, nonspecific esterase positive, and contained large amounts of cytoplasm with one or more nuclei containing several nucleoli per nucleus. Smaller colonies (1 to 2.5 mm in diameter and dense in the center) containing similar cells and making up an additional 14% of the colonies formed in 1 degree wells also showed extensive replating capacity, including generation of larger colonies. These colony-forming cells are likely similar to the murine macrophage high-proliferative potential colony-forming cells. The cells giving rise to these colonies are present in about eightfold higher frequency in cord blood than in adult bone marrow. These cells may at least in part be associated with the successful hematopoietic repopulating capacity of umbilical cord blood cells.

Enrichment, characterization, and responsiveness of single primitive CD34 human umbilical cord blood hematopoietic progenitors with high proliferative and replating potential

To characterize the growth of cord blood progenitor cells, single nonadherent, low-density, T-lymphocyte-depleted CD34 cells were sorted by flow cytometer with an autoclone device into single wells containing culture medium and cytokines. These cells were evaluated for proliferation and for replating ability of their progeny. This latter effect is used as a measure of self-renewal capacity. Colony formation was assessed in 1 degree wells containing various cytokines, alone and in combination, and single colonies deriving after 21 days in semisolid medium were replated into 2 degree wells in the presence of the combination of purified preparations of recombinant human steel factor (SF, a c-kit ligand), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3), and erythropoietin (Epo). Replating of single colonies was performed also for 3 degrees, 4 degrees, and 5 degrees cultures. In the presence of serum, colony formation was observed in &gt; 66% of the wells stimulated with the combination of Epo, SF, GM-CSF, G-CSF, and IL-3, and more than 39% of the colonies formed in these 1 degree wells were very large in size (&gt; 2.5 mm in diameter, dense in the center, and containing &gt; 10(4) cells/colony). The replating efficiency of these large colonies was up to 93% with generation of subsequent colonies of very large size. Replating could be shown for up to five generations. The cells in these colonies were large, nonspecific esterase positive, and contained large amounts of cytoplasm with one or more nuclei containing several nucleoli per nucleus. Smaller colonies (1 to 2.5 mm in diameter and dense in the center) containing similar cells and making up an additional 14% of the colonies formed in 1 degree wells also showed extensive replating capacity, including generation of larger colonies. These colony-forming cells are likely similar to the murine macrophage high-proliferative potential colony-forming cells. The cells giving rise to these colonies are present in about eightfold higher frequency in cord blood than in adult bone marrow. These cells may at least in part be associated with the successful hematopoietic repopulating capacity of umbilical cord blood cells.