Abstract
A xeno-free method for ex vivo generation of red blood cells (RBCs) is attempted in order to replicate for large-scale production and clinical applications. An efficient milieu was formulated using injectable drugs substituting the animal-derived components in the culture medium. Unfractionated mononuclear cells isolated from human umbilical cord blood were used hypothesizing that the heterogeneous cell population could effectively contribute to erythroid cell generation. The strategy adopted includes a combination of erythropoietin and other injectable drugs under low oxygen levels, which resulted in an increase in the number of mature RBCs produced in vitro. The novelty in this study is the addition of supplements to the medium in a stage-specific manner for the differentiation of unfractionated umbilical cord blood mononuclear cells (MNCs) into erythropoietic lineage. The erythropoietic lineage was well established by day 21, wherein the mean cell count of RBCs was found to be 21.36 ± 0.9 × 108 and further confirmed by an upregulated expression of CD235a+ specific to RBCs. The rationale was to have a simple method to produce erythroid cells from umbilical cord blood isolates in vitro by mitigating the effects of multiple erythroid-activating agents and batch to batch variability.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- EPO:
-
Erythropoietin
- UCB:
-
Umbilical cord blood
- MNCs:
-
Mononuclear cells
References
Boehm D, Murphy WG, Al-Rubeai M (2010) The effect of mild agitation on in vitro erythroid development. J Immunol Methods 360:20–29. https://doi.org/10.1016/j.jim.2010.05.007
Brune T, Garritsen H, Witteler R et al (2002) Autologous placental blood transfusion for the therapy of anaemic neonates. Biol Neonate 81:236–243. https://doi.org/10.1159/000056754
Chandrashekar S, Kantharaj A (2014) Legal and ethical issues in safe blood transfusion. Indian J Anaesth 58:558–564. https://doi.org/10.4103/0019-5049.144654
Cheung JOP, Casals-Pascual C, Roberts DJ, Watt SM (2007) A small-scale serum-free liquid cell culture model of erythropoiesis to assess the effects of exogenous factors. J Immunol Methods 319:104–117. https://doi.org/10.1016/j.jim.2006.11.002
Christensen RD, Jopling J, Henry E, Wiedmeier SE (2008) The erythrocyte indices of neonates, defined using data from over 12,000 patients in a multihospital health care system. J Perinatol 28:24–28. https://doi.org/10.1038/sj.jp.7211852
Constantino BT (2011) The red cell histogram and the dimorphic red cell population. Lab Med 42:300–308. https://doi.org/10.1309/LMF1UY85HEKBMIWO
Crauste F, Pujo-Menjouet L, Génieys S et al (2008) Adding self-renewal in committed erythroid progenitors improves the biological relevance of a mathematical model of erythropoiesis. J Theor Biol 250:322–338. https://doi.org/10.1016/j.jtbi.2007.09.041
de Lima M, McMannis J, Gee A et al (2008) Transplantation of ex vivo expanded cord blood cells using the copper chelator tetraethylenepentamine: a phase I/II clinical trial. Bone Marrow Transplant 41:771–778. https://doi.org/10.1038/sj.bmt.1705979
Drake JR, Fitch CD (1980) Status of vitamin E as an erythropoietic factor. Am J Clin Nutr 33:2386–2393
Duran JM, Taghavi S, George JC (2012) The need for standardized protocols for future clinical trials of cell therapy. Transl Res 160:399–410. https://doi.org/10.1016/j.trsl.2012.07.004
England SJ, McGrath KE, Frame JM, Palis J (2011) Immature erythroblasts with extensive ex vivo self-renewal capacity emerge from the early mammalian fetus. Blood 117:2708–2717. https://doi.org/10.1182/blood-2010-07-299743
Garritsen HSP, Brune T, Louwen F et al (2003) Autologous red cells derived from cord blood: collection, preparation, storage and quality controls with optimal additive storage medium (Sag-mannitol). Transfus Med 13:303–310. https://doi.org/10.1046/j.1365-3148.2003.00457.x
Giarratana M-C, Kobari L, Lapillonne H et al (2005) Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells. Nat Biotechnol 23:69–74. https://doi.org/10.1038/nbt1047
Giarratana M-C, Rouard H, Dumont A et al (2011) Proof of principle for transfusion of in vitro-generated red blood cells. Blood 118:5071–5079. https://doi.org/10.1182/blood-2011-06-362038
Giarratana MC, Marie T, Darghouth D, Douay L (2013) Biological validation of bio-engineered red blood cell productions. Blood Cells Mol Dis 50:69–79. https://doi.org/10.1016/j.bcmd.2012.09.003
Glen KE, Workman VL, Ahmed F et al (2013) Production of erythrocytes from directly isolated or Delta1 Notch ligand expanded CD34+ hematopoietic progenitor cells: process characterization, monitoring and implications for manufacture. Cytotherapy 15:1106–1117. https://doi.org/10.1016/j.jcyt.2013.04.008
Haiden N, Klebermass K, Cardona F et al (2006) A randomized, controlled trial of the effects of adding vitamin B12 and folate to erythropoietin for the treatment of anemia of prematurity. Pediatrics 118
Hiroyama T, Miharada K, Kurita R, Nakamura Y (2011) Plasticity of cells and ex vivo production of red blood cells. Stem Cells Int 2011:195780. https://doi.org/10.4061/2011/195780
Icardi A, Paoletti E, De Nicola L et al (2013) Renal anaemia and EPO hyporesponsiveness associated with vitamin D deficiency: the potential role of inflammation. Nephrol Dial Transplant 28:1672–1679
Indumathi S, Harikrishnan R, Rajkumar JS, Dhanasekaran M (2015) Immunophenotypic comparison of heterogenous non-sorted versus sorted mononuclear cells from human umbilical cord blood: a novel cell enrichment approach. Cytotechnology 67:107–114. https://doi.org/10.1007/s10616-013-9663-2
Jin H, Kim H-S, Kim S, Kim HO (2014) Erythropoietic potential of CD34+ hematopoietic stem cells from human cord blood and G-CSF-mobilized peripheral blood. Biomed Res Int 2014:435215. https://doi.org/10.1155/2014/435215
Khodabux CM, von Lindern JS, van Hilten JA et al (2008) A clinical study on the feasibility of autologous cord blood transfusion for anemia of prematurity. Transfusion 48:1634–1643. https://doi.org/10.1111/j.1537-2995.2008.01747.x
Kögler G, Sensken S, Airey JA et al (2004) A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential. J Exp Med 200:123–135. https://doi.org/10.1084/jem.20040440
Lahoti V, Murphy W, Al-Rubeai M (2012) Mathematical approach for the optimal expansion of erythroid progenitors in monolayer culture. J Biotechnol 161:308–319. https://doi.org/10.1016/j.jbiotec.2012.06.003
Lapillonne H, Kobari L, Mazurier C et al (2010) Red blood cell generation from human induced pluripotent stem cells: perspectives for transfusion medicine. Haematologica 95:1651–1659. https://doi.org/10.3324/haematol.2010.023556
Leberbauer C, Boulmé F, Unfried G et al (2005) Different steroids co-regulate long-term expansion versus terminal differentiation in primary human erythroid progenitors. Blood 105:85–94. https://doi.org/10.1182/blood-2004-03-1002
Lim M, Panoskaltsis N, Ye H, Mantalaris A (2011) Optimization of in vitro erythropoiesis from CD34+ cord blood cells using design of experiments (DOE). Biochem Eng J 55:154–161. https://doi.org/10.1016/j.bej.2011.03.014
Lu S-J, Feng Q, Park JS et al (2008) Biologic properties and enucleation of red blood cells from human embryonic stem cells. Blood 112:4475–4484. https://doi.org/10.1182/blood-2008-05-157198
Moore DC, Carter DL, Bhandal AK, Studzinski GP (1991) Inhibition by 1,25 dihydroxyvitamin D3 of chemically induced erythroid differentiation of K562 leukemia cells. Blood 77:1452–1461
Mountford JC, Turner M (2011) In vitro production of red blood cells. Transfus Apher Sci 45:85–89. https://doi.org/10.1016/j.transci.2011.06.007
Neildez-Nguyen TMA, Wajcman H, Marden MC et al (2002) Human erythroid cells produced ex vivo at large scale differentiate into red blood cells in vivo. Nat Biotechnol 20:467–472. https://doi.org/10.1038/nbt0502-467
Pranke P, Hendrikx J, Alespeiti G et al (2006) Comparative quantification of umbilical cord blood CD34+ and CD34+ bright cells using the ProCount-BD and ISHAGE protocols. Braz J Med Biol Res 39:901–906. https://doi.org/10.1590/S0100-879X2006000700008
Ramesh B, Guhathakurta S (2013) Large-scale in-vitro expansion of RBCs from hematopoietic stem cells. Artif Cells Nanomed Biotechnol 41:42–51. https://doi.org/10.3109/10731199.2012.702315
Rosen CJ, Ackert-Bicknell C, Rodriguez JP, Pino AM (2009) Marrow fat and the bone microenvironment: developmental, functional, and pathological implications. Crit Rev Eukaryot Gene Expr 19:109–124. https://doi.org/10.1016/j.bbi.2008.05.010
Rubinstein P, Dobrila L, Rosenfield RE et al (1995) Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution. Proc Natl Acad Sci U S A 92:10119–10122. https://doi.org/10.1073/pnas.92.22.10119
Saraiva BCA, Soares MCC, Dos Santos LC et al (2014) Iron deficiency and anemia are associated with low retinol levels in children aged 1 to 5 years. J Pediatr 90:593–599. https://doi.org/10.1016/j.jped.2014.03.003
Sawada K, Krantz SB, Dessypris EN et al (1989) Human colony-forming units-erythroid do not require accessory cells, but do require direct interaction with insulin-like growth factor I and/or insulin for erythroid development. J Clin Invest 83:1701–1709. https://doi.org/10.1172/JCI114070
Stocchi V, Cucchiarini L, Magnani M, Fornaini G (1987) Adenine and pyridine nucleotides in the erythrocyte of different mammalian species. Biochem Int 14:1043–1053
Taguchi T, Suita S, Nakamura M et al (2003) The efficacy of autologous cord-blood transfusions in neonatal surgical patients. J Pediatr Surg 38:604–607. https://doi.org/10.1053/JPSU.2003.50131
Timmins NE, Nielsen LK (2011) Manufactured RBC - rivers of blood, or an oasis in the desert? Biotechnol Adv 29:661–666. https://doi.org/10.1016/j.biotechadv.2011.05.002
van den Akker E, Satchwell TJ, Pellegrin S et al (2010) The majority of the in vitro erythroid expansion potential resides in CD34(−) cells, outweighing the contribution of CD34(+) cells and significantly increasing the erythroblast yield from peripheral blood samples. Haematologica 95:1594–1598. https://doi.org/10.3324/haematol.2009.019828
Vlaski M, Lafarge X, Chevaleyre J et al (2009) Low oxygen concentration as a general physiologic regulator of erythropoiesis beyond the EPO-related downstream tuning and a tool for the optimization of red blood cell production ex vivo. Exp Hematol 37:573–584. https://doi.org/10.1016/j.exphem.2009.01.007
von Lindern M, Zauner W, Mellitzer G et al (1999) The glucocorticoid receptor cooperates with the erythropoietin receptor and c-Kit to enhance and sustain proliferation of erythroid progenitors in vitro. Blood 94:550–559
Wang J, Kimura T, Asada R et al (2003) SCID-repopulating cell activity of human cord blood-derived CD34- cells assured by intra-bone marrow injection. Blood 101:2924–2931
Xi J, Li Y, Wang R et al (2013) In vitro large scale production of human mature red blood cells from hematopoietic stem cells by coculturing with human fetal liver stromal cells. Biomed Res Int 2013:807863. https://doi.org/10.1155/2013/807863
Xie X, Li Y, Pei X (2014) From stem cells to red blood cells: how far away from the clinical application? Sci China Life Sci 57:581–585. https://doi.org/10.1007/s11427-014-4667-5
Acknowledgments
The authors would like to acknowledge Dr.Asra, Dr. Chandra and Mr. Fida Hussain for their kind help.
Funding
A part of this work is funded by the Department of Biotechnology (DBT), Govt. of India (Ref. No. BT/PR 5729/PID/6/675/2012).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Conflict of interest
The authors declare that they have no conflict of interest.
Electronic supplementary material
Supplementary Fig 1
Cell recovery and purity was determined on samples obtained from the UCB before (pre) and after volume reduction (post) using differential cell counts coupled with flow cytometric analysis of specimens stained with 7-amino-actinomycin D (7AAD) and CD45+ cells. Top panel: FACS plots of unfractionated cord blood (a, b and c). A gate was placed around the population of interest (a) and the events selected were then sent to a 7-AAD /SSC plot to discriminate the live and dead cells (b). The selected even were then sent to a CD45 + /SSC plot, where viable CD45 + cells were counted (c). Bottom panel: FACS plots of MNCs obtained after density gradient centrifugation. The same gating strategy was applied for viable CD45+ cell counting in MNCs fraction (d, e and f). (JPG 436 kb)
Supplementary Fig 2
Flow cytometry results using anti– HbF antibody. The high HbF content of cultured RBCs (~80%) with bright fluorescence, corresponding to HbF, allowed discrimination between day 21 ex vivo generated RBCs (a) and essentially HbF negative adult peripheral blood RBCs (b) . (JPG 41 kb)
Rights and permissions
About this article
Cite this article
Rallapalli, S., Guhathakurta, S., Narayan, S. et al. Generation of clinical-grade red blood cells from human umbilical cord blood mononuclear cells. Cell Tissue Res 375, 437–449 (2019). https://doi.org/10.1007/s00441-018-2919-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00441-018-2919-6