Improving approaches for hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) mobilization is clinically important because increased numbers of these cells are needed for enhanced transplantation. Chemokine stromal cell derived factor-1 (also known as CXCL12) is believed to be involved in retention of HSCs and HPCs in bone marrow. AMD3100, a selective antagonist of CXCL12 that binds to its receptor, CXCR4, was evaluated in murine and human systems for mobilizing capacity, alone and in combination with granulocyte colony-stimulating factor (G-CSF). AMD3100 induced rapid mobilization of mouse and human HPCs and synergistically augmented G-CSF–induced mobilization of HPCs. AMD3100 also mobilized murine long-term repopulating (LTR) cells that engrafted primary and secondary lethally-irradiated mice, and human CD34+ cells that can repopulate nonobese diabetic-severe combined immunodeficiency (SCID) mice. AMD3100 synergized with G-CSF to mobilize murine LTR cells and human SCID repopulating cells (SRCs). Human CD34+ cells isolated after treatment with G-CSF plus AMD3100 expressed a phenotype that was characteristic of highly engrafting mouse HSCs. Synergy of AMD3100 and G-CSF in mobilization was due to enhanced numbers and perhaps other characteristics of the mobilized cells. These results support the hypothesis that the CXCL12-CXCR4 axis is involved in marrow retention of HSCs and HPCs, and demonstrate the clinical potential of AMD3100 for HSC mobilization.

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Summary Hematopoietic stem cells (HSCs) reside in hypoxic niches within bone marrow and cord blood. Yet, essentially all HSC studies have been performed with cells isolated and processed in non-physiologic ambient air. By collecting and manipulating bone marrow and cord blood in native conditions of hypoxia, we demonstrate that brief exposure to ambient oxygen decreases recovery of long-term repopulating HSCs and increases progenitor cells, a phenomenon we term Extra Physiologic Oxygen Shock/Stress (EPHOSS). Thus, true numbers of HSCs in the bone marrow and cord blood are routinely underestimated. We linked ROS production and induction of the mitochondrial permeability transition pore (MPTP) via cyclophilin D and p53 as mechanisms of EPHOSS. MPTP inhibitor Cyclosporine A protects mouse bone marrow and human cord blood HSCs from EPHOSS during collection in air, resulting in increased recovery of transplantable HSCs. Mitigating EPHOSS during cell collection and processing by pharmacological means may be clinically advantageous for transplantation.

We estimated whether single collections of cord blood contained sufficient cells for hematopoietic engraftment of adults by evaluating numbers of cord blood and adult bone marrow myeloid progenitor cells (MPCs) as detected in vitro with steel factor (SLF) and hematopoietic colonystimulating factors (CSFs). SLF plus granulocyte-macrophage (GM)-CSF detected 8-to 11-fold more cord blood GM progenitors [colony-forming units (CFU)-GM] than cells stimulated with GM-CSF or 5637 conditioned medium (CM), growth factors previously used to estimate cord blood CFU-GM numbers. SLF plus erythropoietin (Epo) plus interleukin 3 (IL-3) enhanced detection ofcord blood multipotential (CFU-GEMM) progenitors 15-fold compared to stimulation with Epo plus IL-3. Under the same conditions, bone marrow CFU-GM and CFU-GEMM were only enhanced in detection 2-to 4-and 6-to 8-fold. Increased detection of cord blood CFU-GEMM correlated directly with decreased detection of cord blood erythroid burst-forming units (BFU-E). In contrast, adult bone marrow CFU-GEMM and BFU-E numbers were both enhanced by SLF plus Epo plus IL-3. This suggests that most cord blood BFU-E may actually be CFU-GEMM. Cord blood collections (n = 17) contained numbers of MPCs (especially CFU-GM) similar to the number found in nine autologous bone marrow collections. To assess additional sources of MPCs, the peripheral blood of 1-day-old infants was assessed. However, average concentrations of MPCs circulating in these infants were only 30-46% that in their cord blood. Expansion of cord blood MPCs was also evaluated. Incubation of cord blood cells for 7 days with SLF resulted in 7.9-, 2.2-, and 2.7-fold increases in numbers of CFU-GM, BFU-E, and CFU-GEMM compared to starting numbers; addition of a CSF with SLF resulted in even greater expansion of MPCs. The results suggest that cord blood contains a larger number ofearly profile MPCs than previously recognized and that there are probably sufficient numbers of cells in a single cord blood collection to engraft an adult. Although the expansion data must be considered with caution, as human marrow repopulating cells cannot be assessed directly, in vitro expansion of cord blood stem and progenitor cells may be feasible for clinical transplantation.Circulating blood cells are derived from hematopoietic stem and progenitor cells (1). Bone marrow is the main source of stem and progenitor cells in the adult, but, ontologically, these cells are found first in yolk sac, next in fetal liver and spleen, and subsequently in fetal bone marrow (1-3). Human umbilical cord blood is a rich source of these parent cells (reviewed in refs. 4 and 5). Our previous study (4) inferred that cord blood from single collections should contain enough stem and progenitor cells for hematopoietic reconstitution in a transplant setting, a possibility verified by successful hematopoietic engraftment of children with human leukocyte antigen (HLA)-matched sibling cord blood cells (5-7).The critical question addressed here is whether a single collection ...

Transplanted cord blood (CB) hematopoietic stem cells (HSC) and progenitor cells (HPC) can treat malignant and nonmalignant disorders. Because long-term cryopreservation is critical for CB banking and transplantation, we assessed the efficiency of recovery of viable HSC͞HPC from individual CBs stored frozen for 15 yr. Average recoveries (؎ 1 SD) of defrosted nucleated cells, colony-forming unitgranulocyte, -macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and colony-forming unit-granulocyte, -erythrocyte, -monocyte, and -megakaryocyte (CFU-GEMM) were, respectively, 83 ؎ 12, 95 ؎ 16, 84 ؎ 25, and 85 ؎ 25 using the same culture conditions as for prefreeze samples. Proliferative capacities of CFU-GM, BFU-E, and CFU-GEMM were intact as colonies generated respectively contained up to 22,500, 182,500, and 292,500 cells. Self-renewal of CFU-GEMM was also retained as replating efficiency of single CFU-GEMM colonies into 2°dishes was >96% and yielded 2°colonies of CFU-GM, BFU-E, and CFU-GEMM. Moreover, CD34 ؉ CD38 ؊ cells isolated by FACS after thawing yielded >250-fold ex vivo expansion of HPC. To assess HSC capability, defrosts from single collections were bead-separated into CD34 ؉ cells and infused into sublethally irradiated nonobese diabetic (NOD)͞severe combined immunodeficient (SCID) mice. CD45 ؉ human cell engraftment with multilineage phenotypes was detected in mice after 11-13 wk; engrafting levels were comparable to that reported with fresh CB. Thus, immature human CB cells with high proliferative, replating, ex vivo expansion and mouse NOD͞SCID engrafting ability can be stored frozen for >15 yr, can be efficiently retrieved, and most likely remain effective for clinical transplantation.C ord blood (CB) is a viable alternative to bone marrow for related and unrelated allogeneic hematopoietic stem cell (HSC)͞progenitor cell (HPC) transplantation (1-13). Since our initial preclinical (14-16) and clinical (1, 17-19) studies, there have been Ͼ2,000 CB transplants performed to treat a variety of malignant and nonmalignant disorders in children and adults (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(17)(18)(19). CB HSC͞HPC are frozen before use for transplantation (14,(20)(21)(22), but the longest that a CB collection has been stored frozen before use for clinical transplantation is in the 3-to 5-yr range. With Ͼ100,000 CBs stored frozen world-wide for prolonged periods in anticipation of their clinical use, information on longer-term storage of CB HSC and HPC is of critical importance.The capacity to freeze and retrieve CB HPC cells was first reported when we suggested that CB could serve as a source of transplantable and engrafting HSC and HPC (14). We subsequently evaluated effects of 5-yr (16) and 10-yr (23) storage on retrieval of HPC in which postfreeze HPC numbers were compared directly to prefreeze numbers from the exact same CB samples. Thus, a true recovery rate could be calculated. At those times, we assessed only numbers and proliferation of HPC in vitro. In the present report, we extended anal...

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IntroductionThe first cord blood (CB) transplantation saved the life of a young patient with Fanconi anemia using HLA-matched sibling CB cells, 1 a procedure made possible by identification and cryopreservation of transplantable hematopoietic progenitor cells (HPCs) and hematopoietic stem cells (HSCs) in CB. 2 More than 20 000 CB transplantations have treated the same malignant and nonmalignant disorders as bone marrow (BM). 3-8 CB transplantation is possible because of CB banks, and how long CB can be stored in a cryopreserved state with efficient recovery of HSCs and HPCs is critical for CB banking. We reported highly efficient recovery of CB HPCs after 5, 9 10, 10 and 15 11 years, and recovery of HSCs after 15 years. 11 We now report efficient recovery of functional HPCs up to 21-23.5 years, with more in depth studies on CB HSC engraftment in immune deficient mice, recovery of responsive T cells, generation of induced pluripotent stem (iPS) cells, 12-14 and detection of endothelial colony forming cells (ECFCs). 15 MethodsCB cells were scheduled for discard. 2 The study was approved by the Institutional Review Board of Indiana University (IU). Cryopreservation, thawing, and plating were as reported. 2,9-11 CB was assessed within 36 hours of collection. Cells were either separated into a mononuclear (MNC) fraction (Ficoll-Hypaque; Pharmacia) and aliquoted into cryotubes (Nalge Nunc) or left unseparated and aliquoted into cryo-freezer bags, 2,16,17 in 10% Dimethylsulfoxide and 10% autologous plasma for eventual analysis of HPC recovery. Percent recovery from MNC or unseparated cryopreserved cells was based on total prefreeze cells per volume of the exact same CB unit. 2,9-11 After thaw of unseparated cells, CD34 ϩ cells were magnetic-bead separated 11 for HSC engraftment and iPS cell generation studies. CD4 ϩ and CD8 ϩ T lymphocytes were separated from the CD34 ϩ -depleted cells and stimulated on plates precoated with anti-CD3 (OKT3, 0.5 g/mL) and anti-CD28 (clone CD28.2, 1 g/mL) with 10% FBS, 50M 2ME and 10ng/mL IL-15 as described. 18 Immune-deficient mouse assay for human CB donor chimerism was as reported, 11 except that recipients were NOD/SCID/IL2Rg null (NSG). 19 iPS cell generationAt IU, CD34 ϩ cells isolated from thawed, unseparated cells were grown with 10% FBS, 10 ng/mL human (h) SCF, 10 ng h Flt3-ligand, and 10 ng h Thrombopoietin/mL for 3 days. At day 4, cells were spin-infected (2200 rpm; 45 minutes) with concentrated lentiviral vectors Sox2-Oct4-EGFP and cMyc-Klaf4 (pc DNA-HIV-CS-CGW, provided by Dr P. Zoltick, Children's Hospital, Philadelphia; supplemental Figure 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article) in ␣-MEM medium with polybrene (Sigma-Aldrich). Medium was replaced at 6 days with the cytokines noted in this paragraph. At day 7, cells were transferred to mitotically inactivated murine embryonic fibroblasts (MEFs) and cultured as for human embryonic stem cells (hESCs). 20 iPS cells were also generated at Johns Hopkins using retro...