Hematopoietic stem/progenitor cells, generation of induced pluripotent stem cells, and isolation of endothelial progenitors from 21- to 23.5-year cryopreserved cord blood

Abstract

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...

Cord Blood Transplantation in Adults with Acute Leukemia

“…In fact, clinical trials are now underway in type 1 diabetes, cerebral palsy and peripheral vascular disease. Moreover, recent studies demonstrated that it is possible to generate induced pluripotent stem (iPS) cells from human CB (Giorgetti et al, 2009;Haase et al, 2009;Hu et al, 2011;Broxmeyer et al, 2011). These data offer CB derived iPS cells are also considered an ideal source for future regenerative therapies.…”

Efficient Derivation and Genetic Modifications of Human Pluripotent Stem Cells on Engineered Human Feeder Cell Lines

“…Advantages of using neonatal cord blood cells for generating iPSCs include (1) cord blood may contain a fewer number of acquired somatic mutations compared with other adult cell types from the same individuals; (2) a large number of cord blood samples have already been preserved in cord blood banks. Cord blood samples cryopreserved for more than 20 years have been successfully reprogrammed to iPSCs [48]. In addition, we have previously shown that an episomal vectorbased system efficiently reprogrammed unfractionated cord blood cells without the need for enrichment of progenitor cells [20].…”

Allojenik Kordon Kanı Transplantasyonu

“…Kordon kanındaki hemotopoetik kök hücrelerin engrafman kapasitesinin kemik iliğindekilerden daha fazla olduğu immünosupresif fare deneylerinde gösterilmiştir 19,20 .…”