Blood. 2011 May 5; 117(18): 47734777.
1Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN;
2Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN;
3Department of Medicine, Johns Hopkins School of Medicine, Baltimore, MD; and
4Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN
Received 2011 Jan 12; Accepted 2011 Mar 1.
Cryopreservation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) is crucial for cord blood (CB) banking and transplantation. We evaluated recovery of functional HPC cryopreserved as mononuclear or unseparated cells for up to 23.5 years compared with prefreeze values of the same CB units. Highly efficient recovery (80%-100%) was apparent for granulocyte-macrophage and multipotential hematopoietic progenitors, although some collections had reproducible low recovery. Proliferative potential, response to multiple cytokines, and replating of HPC colonies was extensive. CD34+ cells isolated from CB cryopreserved for up to 21 years had long-term ( 6 month) engrafting capability in primary and secondary immunodeficient mice reflecting recovery of long-term repopulating, self-renewing HSCs. We recovered functionally responsive CD4+ and CD8+ T lymphocytes, generated induced pluripotent stem (iPS) cells with differentiation representing all 3 germ cell lineages in vitro and in vivo, and detected high proliferative endothelial colony forming cells, results of relevance to CB biology and banking.
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).38 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,1214 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.2,911 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.2,911 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 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/IL-2Rgnull (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 -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 (A) 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 (B) and within range for fresh CB.2,912 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 (C) demonstrating retention of immature HPCs.9 Thawed CB contains HPC colonies that can be replated (D), 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.
Recovery of nucleated cellularity, HPCs, HSCs, and immune cells after cryopreservation and long-term frozen storage of CB. (A) Comparative percent recovery of nucleated cells, CFU-GM, and CFU-GEMM compared with prefreeze numbers for 10, 15, and 21-23.5 ...
Using different CB collections cryopreserved as unseparated cells, isolated CD34+ cells efficiently engrafted NSG mice for 6-7 months (E). 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 (F). This demonstrated recovery of functional T-cell subsets.
iPS cells are generated from different cell sources,24,25 including fresh CB,1214 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 (A). iPS cell colonies stained positive for OCT4, NANOG, TRA-1-60, SSEA4, and alkaline phosphatase (B). 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 (C). 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 (D), demonstrates early stages of produced cells. Embryoid bodies developed from iPS cells after removal from MEFs (E), and expressed ectodermal, mesodermal, and endodermal proteins (E). 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 (G). These CB-derived iPS cells were differentiated in vitro (H). 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.
Reprogramming of 21-year-old frozen human CB CD34+ cells to iPS cells (A-H) and recovery of ECFCs (I). The phase contrast images in panels B (FCB-iPS and AP stain), E (top left image), and I were viewed using a Zeiss Axiovert 25 CFL inverted microscope ...
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 (I). 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).
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked advertisement in accordance with 18 USC section 1734.
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: ude.iupui@yemxorbh.
8. Broxmeyer HE, Smith FO. Cord Blood Hematopoietic Cell Transplantation. In: Appelbaum FR, Forman SJ, Negrin RS, Blume KG, editors. Thomas' Hematopoietic Cell Transplantation. 4th ed. West Sussex, United Kingdom: Wiley-Blackwell; 2009. pp. 559576. Sec 4, Chap 39.
16. English D, Cooper S, Douglas G, Broxmeyer HE. Collection and processing of cord blood for preservation and hematopoietic transplantation. In: Areman E, Deeg HJ, Sacher RA, editors. Bone marrow and stem cell processing: A manual of current techniques. Philadelphia, PA: FA Davis Co; 1992. pp. 383384.
Read the rest here:
Hematopoietic stem/progenitor cells, generation of induced ...
- Spinal Disc Treatment IN | Disc Pain Treatment IN | Dr ... [Last Updated On: August 22nd, 2014] [Originally Added On: August 22nd, 2014]
- Indiana Stem Cell Treatment Center [Last Updated On: August 22nd, 2014] [Originally Added On: August 22nd, 2014]
- Indiana University (Stem Cell) - what-when-how [Last Updated On: August 24th, 2014] [Originally Added On: August 24th, 2014]
- Spinal Stem Cell Treatment IN | Spinal Pain Treatment IN ... [Last Updated On: August 26th, 2014] [Originally Added On: August 26th, 2014]
- Bone Marrow and Stem Cell Transplant Treatment | Simon ... [Last Updated On: September 1st, 2014] [Originally Added On: September 1st, 2014]
- Researchers Develop New Cells Meant to Form Blood Vessels, Treat Peripheral Artery Disease [Last Updated On: October 14th, 2014] [Originally Added On: October 14th, 2014]
- IUPUI Stem Cell Research Could Expand Clinical Use of ... [Last Updated On: October 15th, 2014] [Originally Added On: October 15th, 2014]
- Key mechanism, potential target to prevent leukemia found [Last Updated On: November 14th, 2014] [Originally Added On: November 14th, 2014]
- Dr. Nia Smyrniotis at The Miami Stem Cell Treatment Center [Last Updated On: December 24th, 2014] [Originally Added On: December 24th, 2014]
- Stem Cell News | Stem Cell Treatment, Stem Cell Research ... [Last Updated On: December 24th, 2014] [Originally Added On: December 24th, 2014]
- What's the role of virtues in the lab? [Last Updated On: February 3rd, 2015] [Originally Added On: February 3rd, 2015]
- IU researchers create the inner ear from stem cells ... [Last Updated On: February 10th, 2015] [Originally Added On: February 10th, 2015]
- Significant Findings in U.S. National Institutes of Health's Trial of Pluristem's PLX-R18 Cells for Treatment of Acute ... [Last Updated On: February 19th, 2015] [Originally Added On: February 19th, 2015]
- IUPUI biologist receives NIH grant to study how glaucoma develops in stem cells [Last Updated On: March 10th, 2015] [Originally Added On: March 10th, 2015]
- Munster veterinary clinic offers stem cell therapy [Last Updated On: July 2nd, 2015] [Originally Added On: July 2nd, 2015]
- Stem Cell Discoveries - About.com Education [Last Updated On: September 19th, 2015] [Originally Added On: September 19th, 2015]
- Faculty Positions in Stem Cell Biology, Employment | ASCB [Last Updated On: September 30th, 2015] [Originally Added On: September 30th, 2015]
- Ethics of Stem Cell Research (Stanford Encyclopedia of ... [Last Updated On: October 6th, 2015] [Originally Added On: October 6th, 2015]
- StemEnhance Increase Adult Stem Cells from Bone Mirrow [Last Updated On: October 19th, 2015] [Originally Added On: October 19th, 2015]
- Stem Cells and Development - Mary Ann Liebert, Inc. [Last Updated On: October 19th, 2015] [Originally Added On: October 19th, 2015]
- Indiana (Stem Cell) - what-when-how [Last Updated On: July 25th, 2016] [Originally Added On: July 25th, 2016]
- Guidelines for Preventing Opportunistic Infections Among ... [Last Updated On: September 5th, 2016] [Originally Added On: September 5th, 2016]
- The Benefits of Stem Cell Science to Your Health [Last Updated On: November 25th, 2016] [Originally Added On: November 25th, 2016]
- Chris Rickert: Science skeptics shouldn't steer UW hiring - Madison.com [Last Updated On: July 4th, 2017] [Originally Added On: July 4th, 2017]
- Want more stem cells and a higher pain tolerance? Try ... [Last Updated On: July 30th, 2017] [Originally Added On: July 30th, 2017]
- Dance event aims to raise funds for cancer research - Daily Journal [Last Updated On: August 7th, 2017] [Originally Added On: August 7th, 2017]
- Asterias Biotherapeutics Opens Two Additional Clinical Sites for ... - GlobeNewswire (press release) [Last Updated On: August 7th, 2017] [Originally Added On: August 7th, 2017]
- Asterias Enrolls First Patient in Final Cohort of SCiStar Clinical Trial - GlobeNewswire (press release) [Last Updated On: August 9th, 2017] [Originally Added On: August 9th, 2017]
- Washington University School of Medicine; Asterias Biotherapeutics ... - STL.News [Last Updated On: August 25th, 2017] [Originally Added On: August 25th, 2017]
- News | Misericordia University: Alumnus tells first-year students they ... - Misericordia University [Last Updated On: August 25th, 2017] [Originally Added On: August 25th, 2017]
- South Bend man a 'walking miracle' after cancer treatment ... - South Bend Tribune [Last Updated On: September 4th, 2017] [Originally Added On: September 4th, 2017]
- Somatic SNAFUCan a Few Mutant Microglia Cause Neurodegenerative Disease? - Alzforum [Last Updated On: September 4th, 2017] [Originally Added On: September 4th, 2017]
- FDA Cracks Down On Stem-Cell Clinics Selling Unapproved Treatments - Northeast Indiana Public Radio [Last Updated On: September 4th, 2017] [Originally Added On: September 4th, 2017]
- IUPUI researchers finding collaboration, breakthroughs in lab facility - IU Newsroom [Last Updated On: September 6th, 2017] [Originally Added On: September 6th, 2017]
- Regenerative Cell Therapy in Fort Wayne, Indiana [Last Updated On: June 22nd, 2018] [Originally Added On: June 22nd, 2018]
- Stem Cell Treatment Program - Indiana Polyclinic [Last Updated On: July 3rd, 2018] [Originally Added On: July 3rd, 2018]
- Stem Cell Treatment Center | IN [Last Updated On: July 3rd, 2018] [Originally Added On: July 3rd, 2018]
- Learn more about Stem Cell Therapy in Fort Wayne Indiana ... [Last Updated On: October 6th, 2018] [Originally Added On: October 6th, 2018]
- Stem Cell Therapy and Irritable Bowel Syndrome - Indiana ... [Last Updated On: April 5th, 2019] [Originally Added On: April 5th, 2019]
- Stem Cell Research Facilities - Indiana Stem Cell [Last Updated On: May 1st, 2019] [Originally Added On: May 1st, 2019]
- Bone Marrow & Blood Stem Cell Transplant | IU Health [Last Updated On: May 5th, 2019] [Originally Added On: May 5th, 2019]
- A New Vaccine Could Mean the End of Polio - The National Interest Online [Last Updated On: September 22nd, 2019] [Originally Added On: September 22nd, 2019]
- Reviewing Assembly Biosciences Inc. (ASMB)'s and Neuralstem Inc. (NASDAQ:CUR)'s results - CryptoCoinsTribune [Last Updated On: September 22nd, 2019] [Originally Added On: September 22nd, 2019]
- When You Need Stem Cells, You May Appreciate the Donor Registry - 93.1 WIBC Indianapolis [Last Updated On: September 22nd, 2019] [Originally Added On: September 22nd, 2019]
- Research suggests new approach for treating inflammation - ScienceBlog.com [Last Updated On: September 22nd, 2019] [Originally Added On: September 22nd, 2019]
- Juul Temporarily Halts Online Sales Of Flavored E-Cigarettes, But Critics Say That's Far From Enough - Kaiser Health News [Last Updated On: October 21st, 2019] [Originally Added On: October 21st, 2019]
- 15 Antiviral Herbs to Keep You Healthy - Healthline [Last Updated On: October 21st, 2019] [Originally Added On: October 21st, 2019]
- Seeking the Killer Space App with Space Tango - The Planetary Society [Last Updated On: December 3rd, 2019] [Originally Added On: December 3rd, 2019]
- Innovative Cellular Therapeutics Appoints Cell and Gene Therapy Manufacturing Expert Christopher Ballas, Ph.D., as SVP of Manufacturing - BioSpace [Last Updated On: January 9th, 2020] [Originally Added On: January 9th, 2020]
- Robots help them 'find their passion' - News Dispatch [Last Updated On: January 9th, 2020] [Originally Added On: January 9th, 2020]
- Spinal Stenosis and Stem Cell Therapy ... - Indiana Polyclinic [Last Updated On: January 9th, 2020] [Originally Added On: January 9th, 2020]
- Blood drive to honor Franklin child with cancer - Daily Journal [Last Updated On: January 27th, 2020] [Originally Added On: January 27th, 2020]
- If Social Distancing Is Impossible in Prisons, People Should Be Freed - Truthout [Last Updated On: March 20th, 2020] [Originally Added On: March 20th, 2020]
- What Explains The COVID-19 Race Gap? : Shots - Health News - NPR [Last Updated On: April 23rd, 2020] [Originally Added On: April 23rd, 2020]
- Head of FDA urged to ensure any COVID vaccine be free of abortion connection - My catholic standard [Last Updated On: April 23rd, 2020] [Originally Added On: April 23rd, 2020]
- Notre Dame Adjusts to the COVID-19 Pandemic | Notre Dame Magazine | University of Notre Dame - ND Newswire [Last Updated On: July 7th, 2020] [Originally Added On: July 7th, 2020]
- Blocking energy pathway reduces GVHD while retaining anti-cancer effects of T-cells - Science Codex [Last Updated On: November 10th, 2020] [Originally Added On: November 10th, 2020]
- The unsexy side of Covid-19 vaccines gets Wall Streets attention - Deccan Herald [Last Updated On: December 17th, 2020] [Originally Added On: December 17th, 2020]
- Stem Cell Therapy | Indiana Medical Center [Last Updated On: June 23rd, 2021] [Originally Added On: June 23rd, 2021]
- Stem Cell Treatment Centers - Indiana Stem Cell | Call Today [Last Updated On: June 23rd, 2021] [Originally Added On: June 23rd, 2021]
- IU School of Medicine researchers discover new potential for functional recovery after spinal cord injury - Spinal News International [Last Updated On: July 21st, 2021] [Originally Added On: July 21st, 2021]
- Biology for Kids: The Movement of Substances in and out of ... [Last Updated On: December 10th, 2021] [Originally Added On: December 10th, 2021]
- Stem cell laws and policy in the United States - Wikipedia [Last Updated On: December 10th, 2021] [Originally Added On: December 10th, 2021]
- COVID-19 vaccine is a gift that is saving lives - CatholicPhilly.com [Last Updated On: December 24th, 2021] [Originally Added On: December 24th, 2021]
- Diversifying marrow registry critical to achieve medical equity say advocates - WISH TV Indianapolis, IN [Last Updated On: September 16th, 2022] [Originally Added On: September 16th, 2022]
- Experts: Don't believe everyone who is hawking stem cells - The Times of Northwest Indiana [Last Updated On: June 24th, 2024] [Originally Added On: June 24th, 2024]