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

The 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 15¹¹  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 

CB 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⁺ cells were magnetic-bead separated¹¹  for HSC engraftment and iPS cell generation studies. CD4⁺ and CD8⁺ T lymphocytes were separated from the CD34⁺-depleted cells and stimulated on plates precoated with anti-CD3 (OKT3, 0.5 μg/mL) and anti-CD28 (clone CD28.2, 1 μg/mL) with 10% FBS, 50μM 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/IL-2Rg^null (NSG).¹⁹ 

At IU, CD34⁺ 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 α-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 retroviruses expressing Oct4, Sox2, Klf4, and c-Myc.¹²  ECFC assay was performed with MNCs isolated from thawed, unseparated CB.¹⁵ 

We show efficient recovery of HPCs from 23 different collections of MNCs thawed from vials after 21-23.5 years (Figure 1A) compared with the exact same unit's precryopreservation numbers, a recovery similar to 10- and 15-year thaws, that assessed the same plus additional CB units. A range of recoveries was evident, but values were similar whether the same samples were assessed 3 times over 3 years, 2 times over 1-3 years, or twice on the same day (data not shown). Recovery of CFU-GM and CFU-GEMM from unseparated cryopreserved cells (N = 3) was greater than 80% (data not shown), and consistent with recovery from MNCs. It is not clear why some samples resulted in low-efficiency recovery, but assessing the recovery of stored cells by thawing a small sample before their possible use in a clinical transplantation setting could help identify low-recovery CB units, and a decision made as to whether or not to use that unit. Proliferation of HPCs was high (Figure 1B) and within range for fresh CB.^2,9-12  Thawed CB is highly responsive to increased colonies from immature HPCs when GM-CSF plus SCF and/or FL are used to stimulate them, compared with that of only GM-CSF (Figure 1C) demonstrating retention of immature HPCs.⁹  Thawed CB contains HPC colonies that can be replated (Figure 1D), suggesting maintenance of HPCs with limited self-renewal capacity.²¹  Secondary CFU-GM/M colonies formed from single replated CFU-GM/M colonies. CFU-GEMM colonies gave rise to secondary colonies of CFU-GEMM, erythroid progenitors, CFU-GM, and CFU-M.

Using different CB collections cryopreserved as unseparated cells, isolated CD34⁺ cells efficiently engrafted NSG mice for 6-7 months (Figure 1E). In 2 experiments, BM cells from engrafted chimeric mice repopulated secondary mice for 6 months. While we demonstrated engrafting capability of thawed CB after 15 years of storage using first generation NOD/SCID mice,¹¹  those mice did not allow long-term primary engraftment or secondary repopulation. Thus, the current study greatly extends previous findings, and demonstrates recovery of long-term repopulating and self-renewing HSCs. We could not calculate percent recovery of HSCs as this assay was not available when cells were cryopreserved, but this engraftment is similar to fresh CB HSCs.¹¹ 

Attaining vigorous T-cell responses against common viral pathogens is critical for survival after CB transplantation.²²  CB T cells are almost exclusively naive cells, with few effector or memory cells.¹⁸  CB T cells are immature compared with adult T cells because of impaired cytokine production and diminished lytic activity.^22,23  To verify immune capability, CD4⁺ and CD8⁺ T lymphocytes, purified from unseparated CB stored up to 21 years, were activated as assessed by CD3/CD28-induced expression of CD25 (Figure 1F). This demonstrated recovery of functional T-cell subsets.

iPS cells are generated from different cell sources,^24,25  including fresh CB,^12-14  and CB cryopreserved for 5-8 years.^12,14  We generated iPS cells from CB cryopreserved for up to 21 years using Oct4, KLF-4, Sox2, and c-Myc reprogramming with lentiviral vector transduction of CD34⁺ cells at IU (Figure 2A). iPS cell colonies stained positive for OCT4, NANOG, TRA-1-60, SSEA4, and alkaline phosphatase (Figure 2B). Quantitative RT-PCR demonstrated reprogramming via expression of endogenous OCT4, SOX2, and NANOG in comparison to H9 ESC cell line and CD34⁺ cells from which iPS cells were generated (Figure 2C). Unmethylated OCT4 promoter in 2 iPS cell lines generated from thawed CB in comparison to enhanced methylation for CD34⁺ cells from which iPS cell colonies were derived (Figure 2D), demonstrates early stages of produced cells. Embryoid bodies developed from iPS cells after removal from MEFs (Figure 2E), and expressed ectodermal, mesodermal, and endodermal proteins (Figure 2E). Moreover, injection of iPS cell colonies into testis capsules of immune-deficient mice demonstrated teratomas with ectoderm, mesoderm, and endoderm, confirming reprogramming. Generation of iPS cells¹²  at Johns Hopkins with 21-year frozen CB from a different collection produced cells expressing TRA-1-60, SSEA4, NANOG, and OCT4 (data not shown), and produced teratomas¹²  with expression of endoderm, mesoderm, and ectoderm markers (Figure 2G). These CB-derived iPS cells were differentiated in vitro (Figure 2H). Efficiency of iPS cell generation from thawed CB ranged from 0.027%-0.05% per CD34⁺ cell, similar to cultured CD34⁺ cells from freshly isolated or shorter-term frozen CB.¹²  This reprogramming efficiency appears higher than from human adult blood or fibroblastic cells.^12,14  If iPS cells are found to be of clinical utility, which is not yet clear,^24,25  HLA-typed CB stored in banks could serve as a source of such typed cells.

High proliferative ECFCs have been identified in CB.¹⁵  MNCs from thawed, unseparated CB stored frozen for up to 21 years formed ECFC colonies, but their size was smaller than colonies from fresh CB (Figure 2I). ECFC colony numbers from thawed CB (2-5/10⁷ mononuclear cells) were 1/5 to 1/10 numbers from fresh CB, even when colonies from fresh cells versus those frozen and stored for up to 3-6 months were assayed. Thus, the freezing procedure that works well for efficient recovery of HPCs may not be optimal for storage of ECFCs. However, ECFCs that can be cryopreserved and recovered may be of value for regenerative medicine, if clinical applicability is proven.²⁴  Thus, recovery of HSCs, HPCs, and other early cell types bodes well for CB banking and use.

These studies were supported by National Institutes of Health Public Health Service grants NIH R01 HL56416 and NIH R01 HL67384, and a project in NHLBI PO1 HL053586 to H.E.B., NIH R01 HL073781 to L.C., and a grant from the Riley Children's Foundation to M.C.Y. Z.Y. was supported by NIH T32 grant HL007525. The IU production of lentiviral vector was funded by NIH P40RR024928 to K.C., and S.W. is funded on a faculty recruitment grant from NIH (P30 HL101337).

National Institutes of Health

Contribution: H.E.B., M.-R.L., N.P., Z.Y., S.W., K.C., L.C., and M.C.Y. designed experiments; H.E.B., M.-R.L., G.H., S.C., N.P., Y.-J.K., C.M., Z.Y., S.W., K.C., L.C., and M.C.Y. performed research and analyzed and interpreted data; H.E.B. wrote the paper, and H.E.B., M.-R.L., N.P., Y.-J.K., Z.Y., S.W., K.C., L.C., and M.C.Y. edited the paper.

Conflict-of-interest disclosure: H.E.B. is on the Medical Scientific Advisory Board of Corduse, a cord blood banking company. M.C.Y. is a cofounder and consultant to EndGenitor Technologies Inc. The remaining authors declare no competing financial interests.

Correspondence: Hal E. Broxmeyer, PhD, Indiana University School of Medicine, Department of Microbiology and Immunology, 950 West Walnut St, R2-302, Indianapolis, IN 46202-5181; e-mail: hbroxmey@iupui.edu.