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Regenerative Medicine

Red blood cells from pluripotent stem cells for use in transfusion

    ,
    Emmanuel Olivier

    Faculty of Biomedical & Life Sciences, University of Glasgow, University Avenue, Glasgow, G12 8QQ, UK

    ,
    Niove E Jordanides

    Faculty of Biomedical & Life Sciences, University of Glasgow, University Avenue, Glasgow, G12 8QQ, UK

    Scottish National Blood Transfusion Service Cell Therapy Group, Royal Infirmary Edinburgh, 51 Little France Cresent, Edinburgh, E16 4SA, UK

    ,
    Paul de Sousa

    MRC Centre for Regenerative Medicine, University of Edinburgh, Royal Infirmary Edinburgh, 51 Little France Crescent, Edinburgh E16 4SA, UK

    &
    Marc L Turner

    Scottish National Blood Transfusion Service Cell Therapy Group, Royal Infirmary Edinburgh, 51 Little France Cresent, Edinburgh, E16 4SA, UK

    MRC Centre for Regenerative Medicine, University of Edinburgh, Royal Infirmary Edinburgh, 51 Little France Crescent, Edinburgh E16 4SA, UK

    Published Online:https://doi.org/10.2217/rme.10.22

    The use of donated red blood cells in transfusion is a well-established cellular therapy. However, problems including insufficient supply, transfusion-transmitted infections and the need for immunological matching hamper even in the best services. These issues may be eliminated by using pluripotent stem cells to generate universal donor group O, Rhesus D-negative red blood cells. Human embryonic stem cells can be maintained and expanded indefinitely and can, therefore, produce the very large cell numbers required for this application. Red blood cell production is also an attractive goal for pluripotent stem cell-derived therapeutics because it is a well-characterized single cell suspension, lacking nucleated cells and with a low expression of HLA molecules. Much progress has been made; however, a number of challenges remain including scale-up, clinical effectiveness and product safety.

    Papers of special note have been highlighted as: ▪▪ of considerable interest

    Bibliography

    • Leacock JH: On transfusion of blood in extreme cases of haemorrhage. Med. Chir. J. Rev.3,279–284 (1817).
    • Blundell J: An account of obstinate vomiting in which an attempt was made to prolong life by the injection of blood into veins. Med. Chir. Trans.10(Pt 2),296–311 (1819).
    • Schmidt PJ, Leacock AG: Forgotten transfusion history: John Leacock of Barbados. BMJ325,1485–1487 (2002).
    • Landsteiner K: Uber agglutinationserscheinungen normalen menschlichen blutes. Wien Klin. Wochenschr.14,1132–1134 (1901).
    • Loutit JF, Mollisson PL: Advantages of a disodium–citrate–glucose mixture as a blood preservative. BMJ2,744–745 (1943).
    • Rous P, Turner JR: The preservation of living red blood cells in vitro. J. Exp. Med.23,219–248 (1916).
    • Barbara J, Regan F, Contreras M: Transfusion Microbiology. Cambridge University Press, Cambridge, UK (2008).
    • National Blood Data Resource Center: Comprehensive Report on Blood Collection and Transfusion in the United States in 2001. AABB Press, Bethseda, MD, USA (2001).
    • Poel CVD, Janssen M: The collection, testing and use of blood and blood products in Europe in 2003. In: European Health Committee (CDSP) 56th Meeting. Strasbourg, France (2004).
    • 10  Prinoth O: Systems for monitoring transfusion risk. Blood Transfus.6,86–92 (2008).
    • 11  Basran S, Frumento RJ, Cohen A et al.: The association between duration of storage of transfused red blood cells and morbidity and mortality after reoperative cardiac surgery. Anesth. Analg.103,15–20 (2006).
    • 12  Dzik W: Fresh blood for everyone? Balancing availability and quality of stored RBCs. Transfus. Med.18,260–265 (2008).
    • 13  Koch CG, Li L, Sessler DI et al.: Duration of red-cell storage and complications after cardiac surgery. N. Engl. J. Med.358,1229–1239 (2008).
    • 14  Tinmouth A, Fergusson D, Yee IC, Hebert PC: Clinical consequences of red cell storage in the critically ill. Transfusion46,2014–2027 (2006).
    • 15  Zallen G, Offner PJ, Moore EE et al.: Age of transfused blood is an independent risk factor for postinjury multiple organ failure. Am. J. Surg.178,570–572 (1999).
    • 16  Berezina TL, Zaets SB, Morgan C et al.:Influence of storage on red blood cell rheological properties. J. Surg. Res.102,6–12 (2002).
    • 17  Card RT: Red cell membrane changes during storage. Transfus. Med. Rev.2,40–47 (1988).
    • 18  Wolfe LC: The membrane and the lesions of storage in preserved red cells. Transfusion25,185–203 (1985).
    • 19  Gilson CR, Kraus TS, Hod EA et al.: A novel mouse model of red blood cell storage and posttransfusion in vivo survival. Transfusion49(8),1546–1553 (2009).
    • 20  Spahn DR, Waschke KF, Standl T et al.: Use of perflubron emulsion to decrease allogeneic blood transfusion in high-blood-loss non-cardiac surgery: results of a European Phase 3 study. Anesthesiology97,1338–1349 (2002).
    • 21  Tremper KK: Perfluorochemical “red blood cell substitutes”: the continued search for an indication. Anesthesiology97,1333–1334 (2002).
    • 22  Creteur J, Sibbald W, Vincent JL: Hemoglobin solutions 0 not just red blood cell substitutes. Crit. Care Med.28,3025–3034 (2000).
    • 23  Stowell CP, Levin J, Spiess BD, Winslow RM: Progress in the development of RBC substitutes. Transfusion41,287–299 (2001).
    • 24  Winslow RM: Current status of blood substitute research: towards a new paradigm. J. Intern. Med.253,508–517 (2003).
    • 25  Spivak JL, Gascon P, Ludwig H: Anemia management in oncology and hematology. Oncologist14(Suppl. 1),43–56 (2009).
    • 26  Bonig H, Chang KH, Geisen C, Seifried E, Ware C: Blood types of current embryonic stem cell lines are not conducive to culturing “universal-donor” red blood cells. Transfusion48,1039–1040 (2008).
    • 27  Douay L, Andreu G: Ex vivo production of human red blood cells from hematopoietic stem cells: what is the future in transfusion? Transfus. Med. Rev.21,91–100 (2007).
    • 28  Douay L, Lapillonne H, Turhan AG: Stem cells – a source of adult red blood cells for transfusion purposes: present and future. Crit. Care Clin.25,383–398 (2009).
    • 29  Delaney C, Heimfeld S, Brashem-Stein C, Voorhies H, Manger RL, Bernstein ID: Notch-mediated expansion of human cord blood progenitor cells capable of rapid myeloid reconstitution. Nat. Med.16,232–236 (2010).
    • 30  Jiang Y, Jahagirdar BN, Reinhardt RL et al.: Pluripotency of mesenchymal stem cells derived from adult marrow. Nature418,41–49 (2002).
    • 31  D’Ippolito G, Diabira S, Howard GA, Menei P, Roos BA, Schiller PC: Marrow-isolated adult multilineage inducible (MIAMI) cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential. J. Cell Sci.117,2971–2981 (2004).
    • 32  Kucia M, Reca R, Campbell FR et al.: A population of very small embryonic-like (VSEL) CXCR4+SSEA-1+Oct-4+ stem cells identified in adult bone marrow. Leukemia20,857–869 (2006).
    • 33  Zuba-Surma EK, Kucia M, Ratajczak J, Ratajczak MZ: “Small stem cells” in adult tissues: very small embryonic-like stem cells stand up! Cytometry A75,4–13 (2009).
    • 34  McGuckin CP, Forraz N: Potential for access to embryonic-like cells from human umbilical cord blood. Cell Prolif.41(Suppl 1),31–40 (2008).
    • 35  Siegel N, Rosner M, Hanneder M, Freilinger A, Hengstschlager M: Human amniotic fluid stem cells: a new perspective. Amino Acids35,291–293 (2008).
    • 36  Thomson JA, Itskovitz-Eldor J, Shapiro SS et al.: Embryonic stem cell lines derived from human blastocysts. Science282,1145–1147 (1998).▪▪ First description of embryonic stem cell (ESC) isolation from human blastocysts, 17 years after murine ESCs were isolated in 1981.
    • 37  Itskovitz-Eldor J, Schuldiner M, Karsenti D et al.: Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol. Med.6,88–95 (2000).
    • 38  Keller G: Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev.19,1129–1155 (2005).
    • 39  Rao BM, Zandstra PW: Culture development for human embryonic stem cell propagation: molecular aspects and challenges. Curr. Opin. Biotechnol.16,568–576 (2005).
    • 40  Kaufman DS, Hanson ET, Lewis RL, Auerbach R, Thomson JA: Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc. Natl Acad. Sci. USA98,10716–10721 (2001).▪▪ Excellent review of the use of pluripotent stem cells to generate all lineages of the hematopoietic system.
    • 41  Kaufman DS, Thomson JA: Human ES cells – hematopoiesis and transplantation strategies. J. Anat.200,243–248 (2002).
    • 42  Kaufman DS: Toward clinical therapies using hematopoietic cells derived from human pluripotent stem cells. Blood114,3513–3523 (2009).
    • 43  Daley GQ: From embryos to embryoid bodies: generating blood from embryonic stem cells. Ann. NY Acad. Sci.996,122–131 (2003).
    • 44  Kennedy M, D’Souza SL, Lynch-Kattman M, Schwantz S, Keller G: Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures. Blood109,2679–2687 (2007).
    • 45  Kyba M, Daley GQ: Hematopoiesis from embryonic stem cells: lessons from and for ontogeny. Exp. Hematol.31,994–1006 (2003).
    • 46  Snodgrass HR, Schmitt RM, Bruyns E: Embryonic stem cells and in vitrohematopoiesis. J. Cell Biochem.49,225–230 (1992).
    • 47  Wang L, Li L, Shojaei F, Levac K et al.: Endothelial and hematopoietic cell fate of human embryonic stem cells originates from primitive endothelium with hemangioblastic properties. Immunity21,31–41 (2004).
    • 48  Zambidis ET, Park TS, Yu W et al.: Expression of angiotensin-converting enzyme (CD143) identifies and regulates primitive hemangioblasts derived from human pluripotent stem cells. Blood112,3601–3614 (2008).
    • 49  Zambidis ET, Peault B, Park TS, Bunz F, Civin CI: Hematopoietic differentiation of human embryonic stem cells progresses through sequential hematoendothelial, primitive, and definitive stages resembling human yolk sac development. Blood106,860–870 (2005).▪▪ Excellent description of the multistep development of hematopoietic cells as they differentiate from human ESCs. Demonstrates that embryonic developmental processes are recapitulated in vitro.
    • 50  Okita K, Ichisaka T, Yamanaka S: Generation of germline-competent induced pluripotent stem cells. Nature448,313–317 (2007).
    • 51  Takahashi K, Yamanaka S: Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell126,663–676 (2006).▪▪ Seminal discovery that somatic cells can be reprogrammed to become pluripotent by the transfection of four transcription factors.
    • 52  Wernig M, Meissner A, Foreman R et al.: In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature448,318–324 (2007).
    • 53  Takahashi K, Tanabe K, Ohnuki M et al.: Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell131,861–872 (2007).▪▪ The demonstration that reprogramming with four transcription factors works in human cells.
    • 54  Yu J, Vodyanik MA, Smuga-Otto K et al.: Induced pluripotent stem cell lines derived from human somatic cells. Science318,1917–1920 (2007).▪▪ Demonstration that human somatic cells can be reprogrammed using two of the original reprogramming factors but replacing the other two, including c-MYC, with alternative transcription factors.
    • 55  Stadtfeld M, Nagaya M, Utikal J, Weir G, Hochedlinger K: Induced pluripotent stem cells generated without viral integration. Science322,945–949 (2008).
    • 56  Lacoste A, Berenshteyn F, Brivanlou AH: An efficient and reversible transposable system for gene delivery and lineage-specific differentiation in human embryonic stem cells. Cell Stem Cell5,332–342 (2009).
    • 57  Zhou H, Wu S, Joo JY et al.: Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell4,381–384 (2009).
    • 58  Ichida JK, Blanchard J, Lam K et al.: A small-molecule inhibitor of TGF-β signaling replaces sox2 in reprogramming by inducing nanog. Cell Stem Cell5,491–503 (2009).
    • 59  Lin T, Ambasudhan R, Yuan X et al.: A chemical platform for improved induction of human iPSCs. Nat. Methods6,805–808 (2009).
    • 60  Lengerke C, Daley GQ: Autologous blood cell therapies from pluripotent stem cells. Blood Rev.24(1),27–37 (2010).
    • 61  Park IH, Arora N, Huo H et al.: Disease-specific induced pluripotent stem cells. Cell134,877–886 (2008).
    • 62  Feng Q, Lu SJ, Klimanskaya I et al.: Hemangioblastic derivatives from human induced pluripotent stem cells exhibit limited expansion and early senescence. Stem Cells (2010) (Epub ahead of print).
    • 63  Hu BY, Weick JP, Yu J et al.: Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency. Proc. Natl Acad. Sci. USA107(9),4335–4340 (2010).
    • 64  Olsen AL, Stachura DL, Weiss MJ: Designer blood: creating hematopoietic lineages from embryonic stem cells. Blood107,1265–1275 (2006).
    • 65  Brendel C, Neubauer A: Characteristics and analysis of normal and leukemic stem cells: current concepts and future directions. Leukemia14,1711–1717 (2000).
    • 66  Lotem J, Sachs L: Cytokine control of developmental programs in normal hematopoiesis and leukemia. Oncogene21,3284–3294 (2002).
    • 67  Rosenbauer F, Koschmieder S, Steidl U, Tenen DG: Effect of transcription-factor concentrations on leukemic stem cells. Blood106,1519–1524 (2005).
    • 68  Tsiftsoglou AS, Vizirianakis IS, Strouboulis J: Erythropoiesis: model systems, molecular regulators, and developmental programs. IUBMB Life61,800–830 (2009).▪▪ Very comprehensive but accessible description of erythropoiesis both in vitro and in vivo, and during embryo formation.
    • 69  Eilken HM, Nishikawa S, Schroeder T: Continuous single-cell imaging of blood generation from haemogenic endothelium. Nature457,896–900 (2009).
    • 70  Vodyanik MA, Bork JA, Thomson JA, Slukvin II: Human embryonic stem cell-derived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential. Blood105,617–626 (2005).▪▪ First detailed description of multipotent hematopoietic cells generated in co-culture with stroma.
    • 71  Wege H, Le HT, Chui MS et al.: Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential. Gastroenterology124,432–444 (2003).
    • 72  Ng ES, Davis RP, Azzola L, Stanley EG, Elefanty AG: Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation. Blood106,1601–1603 (2005).
    • 73  Olivier EN, Qiu C, Velho M, Hirsch RE, Bouhassira EE: Large-scale production of embryonic red blood cells from human embryonic stem cells. Exp. Hematol.34,1635–1642 (2006).▪▪ First report of large-scale red blood cell production from human ESCs.
    • 74  Qiu C, Hanson E, Olivier E et al.: Differentiation of human embryonic stem cells into hematopoietic cells by coculture with human fetal liver cells recapitulates the globin switch that occurs early in development. Exp. Hematol.33,1450–1458 (2005).
    • 75  Qiu C, Olivier EN, Velho M, Bouhassira EE: Globin switches in yolk sac-like primitive and fetal-like definitive red blood cells produced from human embryonic stem cells. Blood111,2400–2408 (2008).
    • 76  Ma F, Ebihara Y, Umeda K et al.: Generation of functional erythrocytes from human embryonic stem cell-derived definitive hematopoiesis. Proc. Natl Acad. Sci. USA105,13087–13092 (2008).
    • 77  Lu SJ, Feng Q, Park JS et al.: Biologic properties and enucleation of red blood cells from human embryonic stem cells. Blood112,4475–4484 (2008).▪▪ Reported a high degree of expansion during red blood cell production from human ESCs and also demonstrated the functional characteristics of these cells were similar to those from cord blood. Used a process without co-culture that is amenable to scale-up to clinically useful numbers.
    • 78  Klump H, Schiedlmeier B, Baum C: Control of self-renewal and differentiation of hematopoietic stem cells: HOXB4 on the threshold. Ann. NY Acad. Sci.1044,6–15 (2005).
    • 79  Bowles KM, Vallier L, Smith JR, Alexander MR, Pedersen RA: HOXB4 overexpression promotes hematopoietic development by human embryonic stem cells. Stem Cells24,1359–1369 (2006).
    • 80  Kyba M, Perlingeiro RC, Daley GQ: HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors. Cell109,29–37 (2002).
    • 81  Lee GS, Kim BS, Sheih JH, Moore M: Forced expression of HoxB4 enhances hematopoietic differentiation by human embryonic stem cells. Mol. Cells25,487–493 (2008).
    • 82  Unger C, Karner E, Treschow A et al.: Lentiviral-mediated HoxB4 expression in human embryonic stem cells initiates early hematopoiesis in a dose-dependent manner but does not promote myeloid differentiation. Stem Cells26,2455–2466 (2008).
    • 83  Wang L, Menendez P, Shojaei F et al.: Generation of hematopoietic repopulating cells from human embryonic stem cells independent of ectopic HOXB4 expression. J. Exp. Med.201,1603–1614 (2005).
    • 84  Yao S, Chen S, Clark J et al.: Long-term self-renewal and directed differentiation of human embryonic stem cells in chemically defined conditions. Proc. Natl Acad. Sci. USA103,6907–6912 (2006).
    • 85  Chen S, Do JT, Zhang Q et al.: Self-renewal of embryonic stem cells by a small molecule. Proc. Natl Acad. Sci. USA103,17266–17271 (2006).
    • 86  Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH: Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat. Med.10,55–63 (2004).
    • 87  Thomas RJ, Anderson D, Chandra A et al.: Automated, scalable culture of human embryonic stem cells in feeder-free conditions. Biotechnol. Bioeng.102,1636–1644 (2009).
    • 88  Oh SK, Chen AK, Mok Y et al.: Long-term microcarrier suspension cultures of human embryonic stem cells. Stem Cell Res. 2009
    • 89  Phillips BW, Horne R, Lay TS, Rust WL, Teck TT, Crook JM: Attachment and growth of human embryonic stem cells on microcarriers. J. Biotechnol.138,24–32 (2008).
    • 90  Li YJ, Chung EH, Rodriguez RT, Firpo MT, Healy KE: Hydrogels as artificial matrices for human embryonic stem cell self-renewal. J. Biomed. Mater. Res. A79,1–5 (2006).
    • 91  Gerecht S, Burdick JA, Ferreira LS, Townsend SA, Langer R, Vunjak-Novakovic G: Hyaluronic acid hydrogel for controlled self-renewal and differentiation of human embryonic stem cells. Proc. Natl Acad. Sci. USA104,11298–11303 (2007).
    • 92  Siti-Ismail N, Bishop AE, Polak J M, Mantalaris A: The benefit of human embryonic stem cell encapsulation for prolonged feeder-free maintenance. Biomaterials29,3946–3952 (2008).
    • 93  Krawetz R, Taiani JT, Liu S et al.: Large-scale expansion of pluripotent human embryonic stem cells in stirred suspension bioreactors. Tissue Eng. Part C Methods (2009) (Epub ahead of print).
    • 94  Li X, Meng G, Krawetz R, Liu S, Rancourt DE: The ROCK inhibitor Y-27632 enhances the survival rate of human embryonic stem cells following cryopreservation. Stem Cells Dev.17,1079–1085 (2008).
    • 95  Ledran MH, Krassowska A, Armstrong L et al.: Efficient hematopoietic differentiation of human embryonic stem cells on stromal cells derived from hematopoietic niches. Cell Stem Cell3,85–98 (2008).
    • 96  Woll PS, Morris JK, Painschab MS et al.: Wnt signaling promotes hematoendothelial cell development from human embryonic stem cells. Blood111,122–131 (2008).
    • 97  Vijayaragavan K, Szabo E, Bosse M, Ramos-Mejia V, Moon RT, Bhatia M: Noncanonical Wnt signaling orchestrates early developmental events toward hematopoietic cell fate from human embryonic stem cells. Cell Stem Cell4,248–262 (2009).
    • 98  Wilson A, Trumpp A: Bone-marrow hematopoietic-stem-cell niches. Nat. Rev. Immunol.6,93–106 (2006).▪▪ Excellent review of what has been discovered so far regarding the complexity of the hematopoietic niche and its control over stem cells behavior.
    • 99  North TE, Goessling W, Walkley CR et al.: Prostaglandin E2 regulates vertebrate hematopoietic stem cell homeostasis. Nature447,1007–1011 (2007).
    • 100  North TE, Goessling W, Peeters M et al.: Hematopoietic stem cell development is dependent on blood flow. Cell137,736–748 (2009).
    • 101  Manca L, Masala B: Disorders of the synthesis of human fetal hemoglobin. IUBMB Life60,94–111 (2008).
    • 102  Olivieri NF, Weatherall DJ: The therapeutic reactivation of fetal hemoglobin. Hum. Mol. Genet.7,1655–1658 (1998).
    • 103  Bessis M, Mize C, Prenant M: Erythropoiesis: comparison of in vivo and in vitro amplification. Blood Cells4,155–174 (1978).
    • 104  Qiu LB, Dickson H, Hajibagheri N, Crocker PR: Extruded erythroblast nuclei are bound and phagocytosed by a novel macrophage receptor. Blood85,1630–1639 (1995).
    • 105  Zhang J, Ney PA: Autophagy-dependent and -independent mechanisms of mitochondrial clearance during reticulocyte maturation. Autophagy5,1064–1065 (2009).
    • 106  Zhang J, Randall MS, Loyd MR et al.: Mitochondrial clearance is regulated by Atg7-dependent and -independent mechanisms during reticulocyte maturation. Blood114,157–164 (2009).
    • 107  Zhang J, Kundu M, Ney PA: Mitophagy in mammalian cells: the reticulocyte model. Methods Enzymol.452,227–245 (2009).
    • 108  Kundu M, Lindsten T, Yang CY et al.: Ulk1 plays a critical role in the autophagic clearance of mitochondria and ribosomes during reticulocyte maturation. Blood112,1493–1502 (2008).
    • 109  Liu J, Guo X, Mohandas N, Chasis JA, An X: Membrane remodeling during reticulocyte maturation. Blood115(10),2021–2027 (2009).
    • 110  Migliaccio AR, Whitsett C, Migliaccio G: Erythroid cells in vitro: from developmental biology to blood transfusion products. Curr. Opin. Hematol.16,259–268 (2009).
    • 111  James V: Guidelines for the Blood Transfusion Services in the United Kingdom. The Stationary Office, UK (2005).
    • 112  Hiroyama T, Miharada K, Sudo K, Danjo I, Aoki N, Nakamura Y: Establishment of mouse embryonic stem cell-derived erythroid progenitor cell lines able to produce functional red blood cells. PLoS One3,e1544 (2008).
    • 113  Timmins NE, Nielsen LK: Blood cell manufacture: current methods and future challenges. Trends Biotechnol.27,415–422 (2009).
    • 201  Marking Rsearch Bureau www.marketingresearchbureau.com/home2.htm
    • 202  Serious Hazards of Transfusion (SHOT) Annual Report 2008 www.shotuk.org/home/
    • 203  Human Fertilization and Embryology Authority, UK www.hfea.gov.uk
    • 204  Human Tissue Authority, UK www.hta.gov.uk
    • 205  The European Medicines Agency www.ema.europa.eu
    • 206  UK Stem Cell Tool Kit, Department of Health www.sc-toolkit.ac.uk/home.cfm
    • 207  Experimental Medicine Tool Kit, the Medical Research Council www.em-toolkit.ac.uk/home.cfm