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Transfusion Medicine is facing mounting challenges, including but not limited to donor availability, blood supply shortages, and transfusion-associated complications, such as immunogenicity and transmission of viral infections. ‘Blood Pharming’, for in vitro Red Blood Cells (RBC) synthesis, offers a potentially effective approach to addressing the challenges and risks associated with the transfusion of blood and related products. This innovative approach employs cells from variable sources such as Hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPSCs), or immortalized progenitor cell lines, directing their differentiation towards erythropoiesis in an in-vitro environment that mimics the normal bone marrow niche required for erythropoiesis. This review article provides a comprehensive analysis of the progress and hurdles in blood pharming, emphasizing in vitro RBC synthesis for clinical application. In-vitro large-scale production of RBCs offers cutting-edge advantages, such as consistent scalability, the capacity to acquire desired blood phenotypes, and a significant reduction in transfusion-related infections, however, substantial molecular and methodological challenges still need to be addressed before the transfer of this approach from bench to bedside. The review discusses the challenges in ensuring scalability that matches demand and supply, the structural and functional integrity of in-vitro synthesized RBCs compared to their in-vivo counterparts, and the cost-effective methods of RBC synthesis in vitro. It also highlights the importance of implementing thorough characterization and testing protocols to comply with regulatory standards. Additionally, it delves into the ethical concerns associated with commercializing such products. In summary, this review examines the progress and obstacles in the field of in-vitro blood pharming. Through a comprehensive analysis of the present state of the discipline, ongoing scholarly investigations, and prospective avenues of inquiry, our objective is to contribute to a more profound comprehension of the potential impact of synthetic RBCs on the transformation of transfusion medicine

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Frontiers in HematologyOPEN ACCESSEDITED BYKristbjorn Orri Gudmundsson,National Cancer Institute at Frederick (NIH),United StatesREVIEWED BYSophie D. Lefevre,Université Paris Cité, FranceElspeth Payne,University College London, United Kingdom*CORRESPONDENCEHammad Hassanhammad.hassan@aku.eduSheerien Rajputsheerien.rajput@aku.edu†These authors have contributed equally tothis workRECEIVED 19 January 2024ACCEPTED 05 March 2024PUBLISHED 28 March 2024CITATIONHassan H and Rajput S (2024) Bloodpharming: exploring the progress andhurdles in producing in-vitro red bloodcells for therapeutic applications.Front. Hematol. 3:1373408.doi: 10.3389/frhem.2024.1373408COPYRIGHT© 2024 Hassan and Rajput. This is an open-access article distributed under the terms ofthe Creative Commons Attribution License(CC BY). The use, distribution or reproductionin other forums is permitted, provided theoriginal author(s) and the copyright owner(s)are credited and that the original publicationin this journal is cited, in accordance withaccepted academic practice. No use,distribution or reproduction is permittedwhich does not comply with these terms.TYPE ReviewPUBLISHED 28 March 2024DOI 10.3389/frhem.2024.1373408Blood pharming: exploring theprogress and hurdles inproducing in-vitro red bloodcells for therapeutic applicationsHammad Hassan*† and Sheerien Rajput*†Centre for Regenerative Medicine and Stem Cell Research, Aga Khan University, Karachi, PakistanTransfusion Medicine is facing mounting challenges, including but not limited todonor availability, blood supply shortages, and transfusion-associatedcomplications, such as immunogenicity and transmission of viral infections.‘Blood Pharming’, for in vitro Red Blood Cells (RBC) synthesis, offers a potentiallyeffective approach to addressing the challenges and risks associated with thetransfusion of blood and related products. This innovative approach employs cellsfrom variable sources such as Hematopoietic stem cells (HSCs), inducedpluripotent stem cells (iPSCs), or immortalized progenitor cell lines, directingtheir differentiation towards erythropoiesis in an in-vitro environment thatmimics the normal bone marrow niche required for erythropoiesis. This reviewarticle provides a comprehensive analysis of the progress and hurdles in bloodpharming, emphasizing in vitro RBC synthesis for clinical application. In-vitro large-scale production of RBCs offers cutting-edge advantages, such as consistentscalability, the capacity to acquire desired blood phenotypes, and a significantreduction in transfusion-related infections, however, substantial molecular andmethodological challenges still need to be addressed before the transfer of thisapproach from bench to bedside. The review discusses the challenges in ensuringscalability that matches demand and supply, the structural and functional integrityof in-vitro synthesized RBCs compared to their in-vivo counterparts, and the cost-effective methods of RBC synthesis in vitro. It also highlights the importance ofimplementing thorough characterization and testing protocols to comply withregulatory standards. Additionally, it delves into the ethical concerns associatedwith commercializing such products. In summary, this review examines theprogress and obstacles in the field of in-vitro blood pharming. Through acomprehensive analysis of the present state of the discipline, ongoing scholarlyinvestigations, and prospective avenues of inquiry, our objective is to contribute toa more profound comprehension of the potential impact of synthetic RBCs on thetransformation of transfusion medicine.KEYWORDSerythropoiesis, in-vitro RBCs, hematopoietic stem cells, transfusion medicine, bloodsupply shortages, scalabilityfrontiersin.org01Hassan and Rajput 10.3389/frhem.2024.1373408IntroductionBlood transfusion is a cornerstone therapeutic intervention inmedical emergencies and for individuals grappling with conditionssuch as inherited anemias, hemoglobinopathies, myelodysplasias,cancers, and chronic diseases of RBCs. Lifelong transfusion, in somepatients, comes with the increased risk of immunization against thedonated RBCs, which can lead to severe transfusion-relatedcomplications, occasionally culminating in life threateningoutcomes (1–4).The continuous requirement for blood and blood productsrelies primarily on blood donations by volunteers worldwide.According to the World Health Organization (WHO),approximately 118 million blood donations are made annuallyworldwide. The increasing gap in the demand and supply ofblood and blood products is further predicted to exacerbate andpose a global threat to blood services (5). The essential nature oftransfusions is challenged by overwhelming demand for blood,inadequate blood supplies and the potential for adverse reactions,highlighting a significant concern for the global healthcare system(1–4). Numerous efforts are underway to explore alternativeapproaches for ex-vivo generation and expansion of erythrocytesthat may serve as a novel and renewable source of transfusion gradeRBCs (6, 7).In recent years, the field of regenerative medicine has witnessedremarkable strides in the development of alternative approaches totraditional blood transfusions. Among these advancements, theconcept of in-vitro RBC production, often referred to as “bloodpharming,” has emerged as a promising avenue with profoundimplications for therapeutic applications. Blood pharming has ledto the development of protocols that can yield large numbers oflaboratory grown, transfusion grade RBCs termed as“manufactured RBCs” (mRBCs) or cultured RBCs (cRBCs) (4).The in vitro generation of mRBCs has emerged as a vital area oftransfusion research. This review aims to comprehensively explorethe progress in the field and persistent challenges encountered inharnessing the potential of mRBCs for therapeutic application.Concentrated research efforts have pivoted towards using multiplecell sources as starting material, including peripheral blood (PB),cord blood (CB), bone marrow (BM), human adult HSCs,erythroblast progenitor cell lines and iPSCs. Amongst these,iPSCs and erythroblast progenitor cell lines have receivedprominence due to their unparalleled potential for unlimitedexpansion of mRBCs proposing a viable and scalable alternativeto conventional blood transfusions (8). Several protocols are stillunder investigation to explore the possibilities of generatingtransfusion grade mRBCs to overcome the hurdles of scarcity ofdonors, availability of blood for rare blood types and frequencyof transfusions.While we navigate the multifaceted landscape of bloodpharming and mRBC synthesis, a breakthrough in the field oftransfusion medicine was reported through the “RESTORE(Recovery and Survival of Stem Cell Originated Red Cells) trial”,an initiative by the “NHS Blood and Transplant, University ofBristol, National Institute for Health and Care Research CambridgeFrontiers in Hematology 02Clinical Facility”. The trial was aimed to compare the posttransfusion longevity of mRBCs which were developed fromdonor−derived stem cells compared to the standard RBCtransfusion from the same donor (9, 10).As we delve into the intricacies of in-vitro RBC production, it isessential to address not only the scientific achievements but also theethical, regulatory, and translational aspects that govern thetransition of these technologies from the laboratory to the clinic.This review aims to provide a holistic understanding of theprogress achieved and the challenges that lie ahead in realizing thefull potential of in-vitro mRBCs for therapeutic applications. Thisreview aims to navigate the normal physiological process oferythropoiesis, challenging landscape of blood pharming, exploringits scientific underpinnings, current progress, and obstacles still toovercome within the ethical framework and guidelines. For anyoneinterested in the future of healthcare and biotechnology,understanding blood pharming becomes imperative as we enter anew era of transfusion medicine.ErythropoiesisErythropoiesis is a multifaceted process that entails themigration of cells from the embryonic yolk sac to the fetal liver,and eventually to the BM during the course of embryonicdevelopment (11). Within the BM, erythroid progenitor cellsundergo multiple stages culminating in the production ofreticulocytes, which then enter the bloodstream. This process isunder the control of several regulatory factors (12). Erythropoietin(EPO), produced by the kidneys, acts as a stimulatory agent, whilehepcidin, produced by the liver, serves to regulate iron mobilization(13, 14). Key transcription factors, notably GATA binding protein 1(GATA) and Krüppel-like factors (KLF), are integral to theregulation of erythropoiesis (15) (see Figure 1). Any interferencewith these regulatory elements can cause disturbances inerythropoiesis, potentially leading to an overproduction ordeficiency of red cells, or to defects in their morphologicalfunctionality (16).Erythropoiesis takes place within the BM through a series oftightly regulated steps of erythroid progenitor cell differentiation.This process ensures the supply of RBCs, which are essential foroxygen transport. Recent scientific advancements have deepenedour understanding of the molecular underpinnings oferythropoiesis, paving the way for novel treatments forrelated disorders.Erythropoiesis in embryosIn the embryo, erythropoiesis begins in the yolk sac,transitioning to the liver and spleen, and eventually establishingin the BM post-birth (11, 17, 18). Primarily, yolk sac is the site ofprimitive progenitor cells forming blood islands to produceembryonic hemoglobin before birth (19). Erythropoiesis thenmoves to the liver and spleen at around 6–8 weeks of gestation.By the 10–28-week, liver becomes the main site for producing EPO(20, 21). By the second trimester’s end, the BM takes over,frontiersin.orgHassan and Rajput 10.3389/frhem.2024.1373408producing fetal hemoglobin (HbF), which has a high oxygenaffinity, facilitating oxygen transport across the placenta (20).Fetal hematopoiesis is influenced by factors such as ferritin Ironregulatory protein 1 and 2 (IRP1 and IRP2), which regulates irontransfer from the placenta (22), and Erythropoietin receptor(EpoR), which extends through early erythropoiesis (23). In orderto suffice the adaptations to changing oxygen needs after birth, theswitch from fetal liver to BM erythropoiesis facilitates the transitionof fetal globin to adult globin (11).Erythropoiesis in bone marrowBM erythropoiesis involves progenitor cells maturing intoRBCs, with about 20 billion new erythrocytes formed daily (24).Cytokines and growth factors like EPO and stem cell factor (SCF)modulate this process, with the marrow’s microenvironmentproviding additional support (16). Osteoblasts, endothelial cells,and stromal cells within this niche contribute to erythropoiesisregulat ion through cytokine product ion and cel lularinteractions (25).Erythroblast islands in the marrow, with a central macrophagesurrounded by erythroblasts, create a microenvironment essentialfor erythropoiesis. Macrophages provide support with nutrients,adhesion molecules, and phagocytosis of cellular debris (26).Frontiers in Hematology 03Erythropoiesis spans over approximately 14 days, beginningwith HSCs producing megakaryocytes, Burst Forming UnitErythroid (BFU-E), and Colony Forming Unit-Erythroid (CFU-E), and progressing through a series of erythroid precursors toreticulocytes (27, 28). This maturation process involves reduced cellsize and increased hemoglobin content and expression of surfacemarkers (17). Erythroid progenitors, BFU-E and CFU-E, marked byCD34, CD105, CD36, CD71, CD45RA and glycophorin A(CD235a) are abundant in the umbilical cord (UC), BM, and PB(17, 27, 29). Human erythroid precursors show distinctive surfacemarkers at different stages of maturation.Driven by the activity of erythropoietin, erythroblasts divideand undergo a series of cell division, which results in transcriptionaland morphological changes: i.e., degradation of intracellularorganelles, increases hemoglobin content, restructure of cellularmembrane and chromatin condensation which ultimately leads toreduce volume and expulsion of nuclei. Enucleation is essential as ittransforms erythroblasts into reticulocytes, which then mature intoerythrocytes (RBCs). The challenges in replicating this processeffectively in vitro, particularly with iPSCs and immortalized celllines, hinder the large-scale production of functional RBCs fortherapeutic purposes (30, 31).Blood pharming; cell sources for invitro erythropoiesisStudies have employed adult and CB derived HSCs andhematopoietic progenitor cells (HSPCs) to generate RBCs in vitro(32–34) (see Figure 2). However, the accessibility of HSCs and/orHSPCs derived from adult and CB is limited, and the production ofRBCs from these sources is not sustainable (8).Human pluripotent stem cells (hPSCs); including bothembryonic stem cells (ESCs) and iPSCs, offer a promisingFIGURE 2Pathways to Erythropoiesis from Stem Cells: This schematicillustrates the process of generating adult RBCs from various stemcell sources, starting with healthy donors who are selected based onABO blood groups. Pluripotent stem cells can differentiate intoiPSCs or be derived from embryonic stem cells (ESCs), both ofwhich have the capacity to enter the erythroid differentiationpathway. CD34+ HSCs, isolated from these pluripotent sources ordirectly from donors, can be cultured to produce immortalizederythroid progenitor cells, which further differentiate into matureRBCs. The arrow pathways denote the progression fromundifferentiated stem cells to fully differentiated erythroid cells,highlighting the potential for both research and therapeuticapplications in erythropoiesis.FIGURE 1Schematic representation of the regulation of erythropoiesis: Earlyphase of erythropoiesis starts from HSCs and ends with theproerythroblast stage, the late phase of erythropoiesis starts withproerythroblasts to erythroblasts (E) towards enucleation withgeneration of reticulocytes and expulsion of pyrencoytes,maturating into RBCs. The differentiation and maturation of thesecells are regulated by broadly acting hematopoietic cytokines,including erythropoietin (EPO), stem cell factor (SCF) andinterleukin-3 (IL-3) and their receptors (R), the SCF receptor cKIT,EPO receptor EPO-R and IL-3-R, which are required for thebiosynthesis of heme and the production of hemoglobin (Hb). Keyfactors such as BCL11A, GATA1, KLF1 and TAL1 are pivotal in theregulation and development of erythropoiesis. BCL11A plays acrucial role in fetal/adult hemoglobin switch and erythroidprogenitor maturation. GATA1 is essential for the survival anddifferentiation of erythroid lineage cells. KLF1, also known as EKLF, isvital for erythrocyte development and the regulation of severalerythroid-specific genes. TAL1, in conjunction with GATA1,contributes to the control of erythroid and megakaryocyticdifferentiation. These factors collectively orchestrate the complexprocess of erythropoiesis, ensuring the efficient production of RBCs.frontiersin.orgHassan and Rajput 10.3389/frhem.2024.1373408solution by potentially serving as a plentiful source for RBCs owingto their capacity for self-renewal (35–37). The process ofdifferentiating RBCs from hPSCs closely mirrors the key stages oferythropoiesis, in which HSCs and HSPCs are committed togenerating cells of the erythroid lineage under a tightly regulatedmicroenvironment through growth factors and molecules thatmimic the natural phenomenon of erythropoiesis (8).Although cells from these varied sources exhibit similarity tohuman erythrocytes, their therapeutic potential for clinicalapplication is still under investigation.Using HSC or HPSC as starting material forgeneration of RBCsResearch endeavors focusing on the generation of mRBCs dateback to the late 1980s (38–40). Differentiation protocols, involvingCD34+ HSPCs, have been developed and comprehensivelyemployed (8, 41).A widely adopted approach involves a 3-step method of liquidculture of erythroid cells. In the first step of RBC synthesis, HSCsare expanded using EPO, SCF, and interleukin-3 (IL-3) within abase medium such as Iscove’s modified Dulbecco’s medium(IMDM) or serum-free expansion medium. In the second step,erythroblast expansion is induced in the presence of SCF, EPO, andtransferrin. The third step involves subsequent terminaldifferentiation, either with or without EPO. While SCF and EPOare essential for the expansion of erythroblasts, cells in the terminaldifferentiation phase gradually lose receptors for these cytokines.Since enucleated reticulocytes retain ferritin receptors, it isrecommended to supplement transferrin, the supplier of iron toerythroid cells, until the final maturation phase is completed (42).Amongst those, expansion and differentiation of stem cellsharvested from BM, PB and CB, to erythroid lineage wasextensively facilitated through development of a liquid culturesystem (43, 44). Various protocols used for generating RBCs fromCD34+ HSPCs and HSCs harvested from PB, BM and CB have beenextended to hPSCs to determine their RBC differentiation potential(11). These methodologies have been employed and optimizedusing different approaches for step-wise generation of RBCsinvolving varied combinations of essential molecules (45–48).A study by Giarratana et al., demonstrated that approximately90% of CD34+ HSCs and HSPCs harvested from human BM, CB,and PB exhibited characteristics resembling RBCs with the ability ofoxygen transport, post differentiation (49).The significance of microenvironmental factors in the terminaldifferentiation process was effectively demonstrated by the work ofNeildez-Nguyen et al., They developed a culture system utilizingCD34+ HSPCs sources form human CB (50). Although in vitro thisculture system led to a remarkable 200,000-fold increase in thenumber of pure populations of erythroid precursor cells, however,the precursor cells did not undergo terminal differentiation intoRBCs until they were transfused to a mouse model, where presenceof the microenvironment and niche led to complete maturationyielding functional enucleated RBCs.The evidence for involvement of the microenvironment wasfurther supported by the work of Fujimi et al., They used CD34+Frontiers in Hematology 04HSPCs human CB and integrated various approaches to generateRBCs (51). In one of their studies, they utilized human telomerasereverse transcriptase gene (hTERT) stroma (telomerase gene-transduced human stromal cells) of human origin to cultureCD34+ HSPCs. Subsequently, a liquid culture system wasemployed to eliminate the hTERT stroma, followed by co-culturing erythroblasts with macrophages. Compared topreviously employed protocols, a higher rate of enucleation wasobserved using this method.Miharada et al., introduced a further innovation to conventionalprotocols through a feeder cell-free method for efficient enucleatedRBC production, challenging the established notion that cell-to-extracellular matrix adhesion and cell to cell connections in theniche or erythroblastic islands (EBIs) are the primary determinantsof enucleation (52). Their findings confirmed that EBIs alone aresufficient to initiate paracrine factors for efficient enucleation interminal erythropoiesis.While this multi-faceted approach has been proven to beefficient for in vitro RBC production from CD34+ HSPCs ofdiverse origins; however, using HSCs or HPSCs for RBCgeneration faces certain limitations such as challenge in acquiringsufficient sample with minimal batch to batch variation, availabilityof quality CD34+ HSPCs, and compromised enucleation efficiency.Addressing these concerns is imperative to establishing asustainable and reliable source for RBC generation in vitro.Using PSCs as starting material for generationof RBCsThe advent of hPSCs offers a distinct opportunity to address theexisting challenges in terms of the choice of starting material for RBCsynthesis in vitro (8, 36). Comprising of ESCs and iPSCs; hPSCsdemonstrate the ability to regenerate and transform into the threegerm layers including erythroid lineage. A study has shown that bothPB and CB-derived iPSCs are reliable sources for the clinicalproduction of RBCs in vitro. However, PB-derived iPSCs may haveadvantages over CB-derived iPSCs due to the limited availability andlarge amount of CB required for iPSC production (53).Studies have shown that upon inducing erythroid differentiation,PSCs undergo an intermediary phase where they express HSCsmarkers such as CD34 or CD43 (54). Three distinct methodologiesare conventionally employed for the differentiation of iPSCs intoHSCs: a) a co-culture approach involving BM stromal cells, b) a two-dimensional (2D) paradigm implemented on a monolayer cultureplatform, and c) a three-dimensional (3D) strategy revolving aroundthe formation of embryonic bodies (EBs) (55).A study in 2009 validated that co-culturing iPSCs withOP9 cells enhanced their differentiation into CD34 + andCD45 + hematopoietic progenitors, which subsequently gave rise tovarious types of hematopoietic colonies (55). In the presence of OP9mouse BM stromal cells as feeder cells, human hiPSCs exhibit thephenotype of CD34-positive and CD45-positive hematopoietic cells.Tisdale’s group employed a two-step approach where theydifferentiated hPSCs into human yolk sac-like sacs andsubsequently into RBCs using medium containing vascularendothelial growth factor (VEGF), SCF, EPO, thrombopoietinfrontiersin.orgHassan and Rajput 10.3389/frhem.2024.1373408(TPO), Interleukin-3 (IL3), Fms-like tyrosine kinase 3 ligand(FLT3) and Bone Morphogenetic Protein 4 (BMP4) (56, 57).Differentiating hPSCs into hematopoietic stem cells (HSCs) viafeeder cells, monolayer cultures or embryoid body (EB) formationhas been a preferred approach by many researchers. In one suchapproach, human ESCs were expanded in IL3, FLT3 ligand, BMP4,hemin, Insulin- like growth factor 1 (IGF1), insulin and transferrinsupplemented serum free media. Followed by subsequent co-culturewith murine MS5 feeder cells. The protocol generatedhemoglobinized erythroblasts expressing embryonic and fetalglobin in 24 days. However, adult globin was not expressed (58).A similar approach was used by Dias et al., who utilized feeder cellswith a relatively simpler but different cocktail of factors includingcolony-stimulating factor 3 (CSF3), TPO, IL6, EPO, SCF and IL3 fordifferentiation of PSCs into RBCs (59).Dorn et al., established a protocol where CD34+ HSCs weregenerated from EBs and were subsequently differentiated intoenucleated RBCs using a representative cytokine combinationconsisting of SCF, IL3, and EPO (60). Notably, these RBCs wereenucleated and expressed fetal and embryonic hemoglobin.The Kim group pursued erythroid lineage differentiationthrough EB formation with varied factor combinations in twodifferent studies from the same group (61). In the first step, EBswere formed with a cocktail of glycogen synthase kinase 3_ inhibitorVIII (GSK3_ inhibitor VIII), BMP4, VEGF, wingless-relatedintegration site 3A (WNT3A) and activin A. Subsequently,Fibroblast growth factor 1 (FGF1), SCF, and b-oestradiol wereadded for 24 hours followed by 11 days incubation with acombination of BMP4, VEGF, IGF2, FGF1, TPO, heparin, SCF,b-oestradiol, and IBMX to differentiate Embroyed bodies towardsHSC lineage. After 11 days a reduced combo consisting ofhydrocortisone, SCF, IL3, and EPO was used which was furtherreduced to poloxamer 188 to achieve terminal erythropoiesis.The same group utilized another cocktail comprising of VEGF,FGF2, BMP4, WNT2A and activin A for EB formation (62)followed by another cocktail including SCF, EPO, heparin,poloxamer 188, IL3and human serum for terminal differentiationof HSCs to RBCs.Cerdan, Rouleau, and Bhatia explored selective promotion ofhESC differentiation into early erythroblasts through EB formationby adding VEGF to a combination of BMP4, IL6, EPO, IL3, SCF andFLT3 ligand (63). However, these erythroblasts expressed onlyembryonic globin.Another innovative four-step approach was employed by Luet al., aiming to generate RBCs from hESCs (64). Formation andexpansion of hematopoietic cells was aimed in the first two steps byutilizing a cytokine cocktail and serum free medium wassupplemented with FLT3 ligand, TPO, FGF2, SCF, BMP4, VEGFand triple protein transduction domain-homeobox B4 (tPTD-HoxB4) fusion protein. Differentiation and enrichment of RBCswas achieved in the third and fourth steps where only SCF and EPOwere added to the medium. The protocol yielded adult globinexpressing enucleated RBCs. This was the first studydemonstrating generation of RBC with normal physiologicalfunction where comparable oxygen equilibrium curves wereobserved in PSC-derived RBCs and adult RBCs.Frontiers in Hematology 05To address challenges associated with culture-to-culturevariability and labor intensity, some protocols have adoptedmonolayer culture systems without EB formation (65–67).Using immortalized cell lines as starting materialfor generation of RBCsUtilizing immortalized erythroid progenitor cell lines for RBCsynthesis is another approach. Notably, Akimov et al., generated acord blood CD34+ cell line through lentiviral co-transduction ofHPV16 E6, E7, and hTERT, extending the cell line’s lifespanwithout tumorigenic activity (54).Kurita et al., established Human iPS-TAL1 cell-derivederythroid progenitor (HiDEP) and human umbilical cord blood-derived erythroid progenitor (HUDEP) cell lines derived fromhuman iPS cells and umbilical cord blood CD34+ HSCs,respectively. The lines showed erythroid differentiation and geneexpression similar to in vitro -cultured CB erythroid cells (68).Additionally, Trakarnsanga et al. (69) developed BristolErythroid Line - Adult (BEL-A) from BM CD34+ cells transducedwith HPV16 E6/E7, resulting in up to 30% enucleated cells afterdifferentiation. The cell line closely resembled normal adulterythropoiesis by expressing surface markers akin to control cellsand predominantly synthesizing hemoglobin A (HbA) (69). Thedifferentiated cells lacked nuclei, thereby eliminating thetumorigenic potential of oncogene-mediated immortalization.More recently, detailed characterization of these cell lines hasfurther shown that they accurately recapitulate their primary cellequivalents, thereby providing a molecular signature forimmortalization (70).Wong et al. (71) used three lentiviral factors SV40 large Tantigen (SV40T), hTERT, and HPV16-E6/E7 genes to immortalizeCD36+ erythroid progenitor cells to generate immortalized CD36+erythroblast (CD36E) cell line. HPV16-E6/E7 expression, alone orwith hTERT, increased CD36+ EPC proliferation, resulting inelevated hemoglobin-producing cells (27% increase) and a shiftfrom HbA to HbF. However, this transformation led tochromosomal alterations, loss of CD34, and changes in geneexpression favoring lymphoid-associated factors over erythroiddifferentiation markers (71, 72).Using peripheral blood mononuclear cells asstarting material for generation of RBCsHeshusius et al., utilized peripheral blood mononuclear cellswithout prior CD34+ isolation (73). They developed a GMP-grademedium and erythroid culture protocol which resulted in over 90%enucleation of RBCs. This method holds great potential for cost-effective large-scale production of RBCs. Notably, the expandederythroblast cultures differentiated into mature RBCs, and theirdeformability and oxygen-binding capacity were comparable to invivo reticulocytes.Present research endeavors also focus on replicating the BMmicroenvironment using 3D culture systems that simulateoptimum conditions for erythroid differentiation. Severn et al.,introduced porous polyurethane scaffolds for PB CD34+ cellinoculation, demonstrating superior HPC egress compared tohuman bone scaffolds (74). Erythroid progenitor proliferation onfrontiersin.orgHassan and Rajput 10.3389/frhem.2024.1373408polymerized high internal phase emulsion (polyHIPE) scaffolds,mimicking the structural characteristics of human BM, resulted insignificantly augmented cell density and erythroid cell production.Lee et al., investigated microporous micro-carriers for erythroid cellculture. Larger diameter carriers facilitated increased cell densityand high enucleated RBC yields, whereas smaller pore sizes limitedcell interaction, leading to reduced cell viability (75).Hence, diverse strategies have been employed for generatingRBCs from different starting materials, exploring a range ofdifferentiation approaches, factor combinations, and culturesystems, however, much remains to be explored to effectivelyassess their therapeutic potential for clinical application.Current challenges and future directionsThe ultimate objective of generating transfusion grade RBCsposes several key challenges, such as achieving effective terminaldifferentiation and enucleation, sustainability, increased yield,scalability, and cost efficiency (31, 73). These factors arediscussed briefly:Concerns regarding cell of originThe initial research on mRBCs primarily focused on humanHSCs. Despite demonstrating multiple sources of erythroidprecursors capable of terminal differentiation with varyingdegrees of efficiency, the prevailing methods rely heavily onutilizing primary HSCs extracted from BM, CB or PB. Thisinvolves expanding a specific subset of stem cells, initiallyenriched through CD34+ cell isolation or direct expansion fromperipheral blood mononuclear cells, by employing a combination ofgrowth factors.These processes allow for the generation of a large quantity ofproerythroblasts, which can then undergo differentiation toproduce enucleated reticulocytes, resembling young RBCs. Theseartificially produced reticulocytes display similar traits to thosederived in vivo, undergo maturation into biconcave erythrocytespost-transfusion, and have shown promising circulatory lifespans inearly-stage human clinical trials (31).Despite success in generating enucleated RBCs, these cells tendto generate fetal hemoglobin (Hb) which has a greater propensity todenature and cause membrane damage in contrast to adult Hb.Moreover, there are limitations to their long-term proliferation andthe necessity for repeated transductions in successive cultures fromdifferent donor sources (31).The process of generating RBCs from hPSCs mirrors the keydevelopmental stages of erythropoiesis, involving the commitmentof HSCs and/or HSPCs to the erythroid lineage (8). However,achieving efficient differentiation of HSPCs from iPSCs remains asubstantial challenge. Equally significant is the establishment of astandardized protocol to guide HSPC differentiation towardserythroid lineages. Despite these advancements, these cells exhibitlimited enucleation potential and minimal expression of adulthemoglobin, if any.Frontiers in Hematology 06Mass scale production of transfusion grade RBCsThe primary challenge in mRBC synthesis lies in achieving aproduction scale to meet standard therapeutic transfusion doses foradults, necessitating over 2×1012 RBCs per unit (42). However,currently employed culture protocols have the potential to generate3-50 units of packed RBCs from one cord blood donation.Optimized culture conditions are required to mitigate thischallenge, particularly ensuring > 90% enucleation for higheryields of adult hemoglobin-expressing erythrocytes, accuratelyexpressing blood group antigens, such as O Rh-negative, andestablishing a universal blood type (72). Furthermore, the primaryhurdle in clinical application lies in the efficacy of differentiationprotocols. Existing methodologies fall short in generating amplefunctional RBCs due to ineffective RBC differentiation. Terminalmaturation issues, especially sustained b-globin expression andenucleation, hinders the process of mRBC synthesis in vitro.Besides quantity, the focus extends to functional and uniformRBC populations, urging the need for extensive research andidentification of robust protocols (8).The use of 3D culture methods and bioreactors is beingexplored to overcome the challenge and to improve themanufacturing efficiencies of mRBCs. This approach includesleveraging different cell sources, bioreactors, and 3-dimensionalmaterials. However, there’s a need for further research to optimizethese methods for clinical application (42). Heshusius et al.,engineered a scalable culture methodology utilizing a G-Rexbioreactor, resulting in the generation of pure erythroid culturesfrom PBMCs without prior CD34+ isolation with a 3x107-foldincrease in erythroblast production within 25 days (73).Remarkably, the cultured RBCs showed a strong functional andmorphological resemblance to in vivo RBCs. However, the costinvolved in RBCs synthesis through bioreactors makes it unlikelythat mRBCs can be produced in clinically required numbers in nearfuture. Significant advances are required in protocols forerythroblast expansion and bioreactor technologies to achievesufficient production of mRBCs.Almost all the methods currently employed for RBC synthesishave challenges such as culture heterogeneity and complex-laborintensive protocols. Establishing adaptable protocols which willyield consistent product and functionally relevant RBCs isindispensable for clinical application of this innovativetechnology. However, further understanding of these techniquesis still in process (4).Cost evaluationIndustrial preparation of mRBC synthesis for clinicalapplication is expensive and complex. A single unit of bloodcontaining approximately 2 X 1012 RBCs costs around US$200-300. However, it is estimated that mRBCs would be more expensive,costing approximately between US$1000-$15,000 for the samenumber of cells. Several variables contribute to the highproduction costs of mRBCs. One such significant factor is thechoice of starting material. The process of culture, expansion anddifferentiation to RBCs is undertaken in vitro with the help offrontiersin.orgHassan and Rajput 10.3389/frhem.2024.1373408certain commercial media components and growth factors that mayincrease the cost significantly. The cost may also increase significantlybecausemultiple batches and flasks would be required to produce thislarge number of cells available for transfusion. Additionally,structural, and functional characterization of these mRBCs againinvolves commercial products and services that will increase theoverall production expense of the product, hence increasing the costof the product (3).Current research is also emphasized in the development of cost-effective protocols for this process. Several studies are focusing oncreating an “economically friendly” protocol, aiming to minimizethe reliance on expensive recombinant growth factors, especiallyerythropoietin (EPO), or exploring alternative strategies to reducecosts. Considering the potential need for large-scale production ofRBCs for future clinical applications, the development of protocolsthat minimize costly resources holds significant importance (55).Methods of reprograming; integration method/non-integrating method/small moleculesCellular reprogramming of somatic cells to a pluripotent staterequires expression of specific transcription factors (TFs) termed asOSKM factors. The famous OSKM factor combination, consistingof Oct4, Sox2, Klf4, and c-Myc, was demonstrated by Yamanaka’slab (76). Although varied combination for reprogramming havebeen identified, OSKMs factors are commonly used forreprogramming. While further research is ongoing to determinethe precise mechanisms through which somatic cells transitionfrom their mature state to a pluripotent state (See SupplementaryTable 1).Reprogramming techniques have advanced concurrently ourunderstanding of transcription factors. Initially, viral integratingtechniques were used to achieve TF expression, but these methodsare not suitable for clinical therapeutics (3).Advancements in lentiviral vector design have been pursuedwith the aim of enhancing both safety and efficiency; however,persisting challenges include the risks associated with off-targeteffects, immune responses, and the potential for randommutagenesis (77–81). Despite improving the methodology,concerns regarding insertional mutagenesis persist, as lentiviralintegration may activate oncogenes or silence tumor suppressorgenes, fostering clonal expansion and the development ofmalignancies (81). Ongoing investigations continue to scrutinizethe integration profile of lentiviral vectors in efforts to mitigate theserisks. Additionally, the phenomenon of off-target effects poses asignificant concern, as unintended vector integration into thegenome may disrupt gene function or regulatory networks,potentially culminating in cellular dysfunction or neoplastictransformations (80). The introduction of genetically modifiedcells into the body may incite immune responses, leading to therejection of transduced cells or systemic immunological reactions, acritical consideration particularly in the context of chronicadministration of lentivirally-derived red blood cells fortherapeutic purposes (77). Ensuring efficient transduction anddirected differentiation of stem cells into fully functional redblood cells presents another obstacle, with inefficienciespotentially compromising the yield and functionality of theFrontiers in Hematology 07resultant cells, thereby impacting therapeutic efficacy (M.-C. 78).Moreover, the ethical and regulatory landscape surrounding theuse of genetic modification technologies in human subjectsnecessitates careful consideration, encompassing informedconsent procedures, ethical concerns regarding genetic alterations,and the negotiation of regulatory approvals, all of which areimperative for the advancement of lentiviral red blood cellgeneration toward clinical applications (79). To avoid aboementioned challenges associated with lentiviral ectors researchershave been working on developing non-integrative techniques toexpress reprogramming factors.Another approach for immortalizing erythroid cells primarilyinvolves utilizing human papillomavirus (HPV)-based in vitrosystems. These lines require stringent assays aimed at detectingany residual nucleated cells in the final product, a necessity for allerythroid cultures from diverse stem cell sources (69, 72)(see Figure 3).Using red blood substitutesRed blood cell substitutes are being developed as an alternativeto blood transfusions. These substitutes aim to mimic the oxygen-carrying capability of blood and are designed to behave similarly tonatural blood in microcirculation. There are two main types of redblood cell substitutes: hemoglobin-based oxygen carriers (HBOCs)and perfluorocarbon-based oxygen carriers (PFCs). However, nooxygen-carrying blood substitutes have been approved for use bythe US FDA due to side effects and short half-life. Scientists areactively working to overcome these challenges and develop realblood substitutes, such as red blood cells obtained throughdifferentiation of stem cells (82, 83). Research on artificial bloodsubstitutes has focused on creating alternatives for specificfunctions of blood, particularly oxygen transport by red bloodcells and hemostasis by platelets. Hemoglobin-based substitutesuse hemoglobin from various sources, while perfluorocarbon-basedFIGURE 3Comparative Analysis of Globin Expression in Different Cell Sources:This diagram presents the proportion of a-globin, b-globin, and g-globin expression across various cell types. ‘PB’ representsperipheral blood, ‘BM’ stands for bone marrow, and ‘CB’ indicatescord blood, each exhibiting a unique composition of globin chains.In addition to these primary sources, the graph also comparesglobin expression in immortalized erythroid cell lines and iPSCs. Thered segment denotes the presence of a-globin, the blue segmentrepresents b-globin, and the yellow segment indicates g-globin. Thisdistribution provides insights into the globin expression profiles andthe potential use of these cells in therapeutic applications orresearch studies related to hemoglobinopathies and otherblood disorders.frontiersin.orgHassan and Rajput 10.3389/frhem.2024.1373408substitutes are completely synthetic compounds. These substituteshave shown increased safety levels, do not require blood typing, anddo not seem to cause immunosuppression in recipients. Despite theprogress, current red cell and platelet substitutes have a shortduration of action and can interfere with clinical laboratorytesting (83, 84). The development of a universal blood substituteremains elusive despite numerous attempts over the years. Whilethere have been advancements in hemoglobin-based oxygencarriers showing promise in early clinical trials, no FDA-approved products are available yet. Challenges in developingblood substitutes include the need for compatibility testing withdonor blood, sterilization methods, and ensuring efficient oxygentransport to tissues.In summary, the quest for effective bloodsubstitutes continues as researchers strive to overcome existingchallenges and develop safe alternatives to traditional bloodtransfusions (85).Structural and functional integrity of in-vitrosynthesized RBCsDue to the increasing demand for safe alternatives to bloodtransfusions, there has been significant interest in mRBCs synthesisin vitro. However, before implementing this innovative idea inmedical practice, mRBCs need thorough evaluation as they mustnot only mirror the morphological properties such as enucleatedcells with biconcave shape switching hemoglobin and a particularmembrane morphology of natural RBCs but also mimic thebiological functions of their natural counterparts includingimmunological response, exchange of gases, oxygen-carryingcapacity, and lifetime of mRBCs.The biconcave structure provides sufficient area for gaseousexchange and provides flexibility to the cells to pass through narrowblood capillaries (86). Since extensive research is focused ongenerating functional mRBCs, we have a limited number ofstudies where the ultimate objective has been achieved. Efforts arestill underway to completely understand the molecular details of theunderlying process and to achieve it in vitro.Enucleation is a key characteristic feature indicating terminaldifferentiation of mature RBCs. However, mRBC synthesis is largelyhampered by the low enucleation efficiency of cultures in vitro andthe maximum enucleation efficiency as reported by research groupsaround the world has been 42% (8). Studies of the molecular detailsto understand the enucleation process are at their early stages.Stable expression of b-globin is amongst the biggest challengesin generating transfusion-grade mRBCs. During the process ofterminal differentiation of enucleated RBC, embryonic and fetalglobin are naturally repressed, and expression of b-globin iskickstarted in adult RBCs. Although extensive efforts have beenmade to overcome the challenge of b-globin switch in mRBCs, thefield is still an area of active investigation (8).The oxygen-carrying capacity, longevity in circulation, andimmunogenicity are key structural and functional characteristicsthat mRBCs must possess to be deemed appropriate for transfusionas RBCs. Although significant efforts have been made in the area, itis still a subject of ongoing research.Frontiers in Hematology 08Ethical concerns associated with thecommercialization of RBC productsBlood pharming offers a promising avenue not only for substitutingdonor blood products but also for potentially augmenting thetherapeutic efficacy of these blood cells through genetic modification.However, universally recognized ethical concerns regarding theutilization of human stem cells (hSC), coupled with subsequentadjustments in legislative regulations, significantly restrict theapplication of ESCs in research. Similarly, generating pluripotentstem cell lines from oocytes and embryos triggers the controversyregarding the definition of human personhood (87).Conversely, ethical considerations surrounding ESCprocurement have impeded research progress, steering attentiontoward alternative resources. iPSCs, introduced by ShinyaYamanaka and Kazutoshi Takahashi, offer an ethicallyuncomplicated alternative resembling ESC properties. iPSCs havebecome a primary focus in developing efficient and scalableprotocols for blood cell production as they possess similarcharacteristics to ESCs with minimal or conditional ethicalconcerns. Successful reports have demonstrated the derivation ofRBCs from iPSCs, showcasing terminal maturation and enucleationin vitro (8, 59, 88). However, stringent regulations are required forbedside applications of iPSCs. These regulations include ethical,legal, and social considerations of cellular therapy including but notlimited to (i) extensive characterization of clinical-grade cells for theabsence of potential of acquiring secondary disease-causingmutations, (ii) adherence to GMP grade manufacturingapplications, (iii) genetic manipulations of cells (89).Umbilical cord blood shows great promise to produce RBCs invitro. Cord blood cells, typically discarded after birth, offer a readilyavailable source. With proper consent, utilizing umbilical cordblood avoids complicating critical or ethical concerns, presentinga valuable resource for RBC production (90).Clinical trialsThe “RESTORE (Recovery and Survival of Stem Cell OriginatedRed Cells) trial” is a pioneering effort in transfusion medicine byintroducing a novel approach involving the infusion of laboratory-cultivated RBCs into recipients requiring blood transfusions. It is arandomized control trial by the “NHS Blood and Transplant,University of Bristol, National Institute for Health and CareResearch Cambridge Clinical Facility,” in collaboration with otheruniversities (10).The ultimate goal of the trial is to be able toproduce rare blood types. The trial involves transferring laboratory-grown RBCs into an individual requiring blood transfusion (9).Donor derived stem cells were differentiated to RBCs followedby transfer of these cells to volunteers. The methodology of trial isbased upon a culture system established by Cogan et al. and Griffithet al. where mature reticulocytes were produced in 20 days fromCD34+ adult PB derived cells (91, 92). The methodology delineatedin the trial manuscript lacks explicit clarity; however, pertinentfrontiersin.orgHassan and Rajput 10.3389/frhem.2024.1373408protocol details are inferred through referenced sources suggestingthat a three-stage method of Douay and Giarratana was used toculture the cells. IMDM containing AB Serum, fatal bovine serum,Insulin, heparin and Transferrin based media was used for culture.The media was supplemented with SCF, IL-3 and EPO in the firststage (days 0-8). During the second stage (days 8-11) SCF, EPO andiron saturated transferrin were supplemented followed by the finalstage (days 11-20) where cells were incubated with mediacontaining EPO and 800mg/ml iron saturated transferrin. After 20days, mature reticulocytes and erythrocytes were isolated andcultured without the use of feeder layers where up to 90%enucleation efficiency was seen. The cells contain normalglycosylation profile and expressed adult hemoglobin withcomparable capacity to oxygen,The life span of this mini blood transfusion (10 ml) during thetrials will be compared to the RBC injection with an equal quantityof regular RBCs obtained from the same donor. The volunteersreceived two mini transfusions with a gap of 4 months. The study isstill ongoing. Volunteer’s blood sample will be collected after sixmonths of the first injection of lab grown RBCs and will be testedfor the presence of tracer labeled laboratory synthesized RBCs. Thetrial holds high expectations in the scientific community as it offersseveral advantages: it will offer an opportunity to synthesize bloodfor rare blood types, hence lifting the requirement of blood donorsfor every patient. It may reduce the frequencies of bloodtransfusions. Although this one trial may not answer all ourquestions, it can be a breakthrough scientific innovation and willfacilitate future endeavors in the field.ConclusionThis comprehensive review endeavors to delve into the currentadvancements and persistent challenges within the domain of ex-vivo synthesis of functional and clinically viable mRBCs. Thepursuit of artificial substitutes for RBCs commenced nearly half acentury ago, yet attaining a successful outcome has remainedelusive, with no immediate substitute yet. This protracted questunderscores the intricate nature of in vitro RBC culture,underscoring the complexities inherent in replicating blood tissueand the existing technological constraints.The identification of HSCs, iPSCs, immortalized erythroid cellsand ESCs offered a significant breakthrough to varied areas inregenerative medicine approach including mRBC synthesis.Research initiatives have diversified into various cell sources asdiscussed in this review. Particularly noteworthy are iPSCs anderythroblast progenitor cells, which have gained prominence due totheir unparalleled capacity for unlimited expansion, representing atransformative stride toward a viable and scalable alternative totraditional blood transfusions. As we navigate the complexities of invitro RBC production, this review amalgamates insights fromdiverse studies, encapsulating advancements in stem cell biology,bioprocessing techniques, and biomaterial engineering. Thesecollective efforts contribute to the intricate process of generatingRBCs outside the human body.Frontiers in Hematology 09Ex vivo expansion of erythrocytes is a potential approach for RBCsynthesis for the last few years. The review by Migliaccio et al.provides an in-depth analysis of the current landscape surroundingthe ex vivo expansion of red blood cells (RBCs) and its potential forclinical transfusion applications in humans. (93). The authorsextensively explore the methodologies employed in RBC expansion,emphasizing the critical role of identifying proper sources of HSCsand optimizing culture conditions to support efficient erythropoiesis.They discuss the diverse strategies utilized for stimulating HSCdifferentiation into mature RBCs, including the use of growthfactors, cytokines, and various culture systems. The potentialclinical implications of ex vivo expanded RBCs are extremelyrelevant in scenarios where conventional blood transfusions are notfeasible, such as in patients who refuse allogeneic transfusions due toreligious beliefs, as in the case of Jehovah’s Witnesses. However, theex vivo expansion of RBCs still faces significant challenges intransfusion medicine such as the concerns related to the safety,scalability, and cost-effectiveness of the expansion process. Theapplication of this technique needs continued research effortsaimed at addressing these challenges and optimizing ex vivo RBCexpansion technologies for practical clinical use.Ongoing basic research is constantly setting the groundwork fortransitioning scientific information to industrial-scale production,thereby facilitating clinical trials with an objective to establish anindustrial technology for the manufacturing of mRBCs, analogous tothe mass production of pharmaceuticals, hence “blood pharming”.This necessitates the establishment of a prototype for large-scaleproduction under pharmaceutical-grade conditions, not only forconducting clinical trials but also to lay the foundation for broaderapplications of this transformative technology. Each milestoneachieved on this road has heightened interest and research in thisfield, expanding the horizons of possibilities for stem cell technologyand its practical applications within the ethical framework.This perspective underscores a pivotal challenge in regenerativemedicine developing large-scale bioengineering solutions forclinical trials to substantiate the efficacy of novel approaches. Theaspiration is that such advancements, including the massproduction of cultured RBCs, will soon become a reality.Author contributionsHH: Writing – original draft, Writing – review & editing,Conceptualization. SR: Conceptualization, Writing – originaldraft, Writing – review & editing.FundingThe author(s) declare financial support was received forthe research, authorship, and/or publication of this article. Theproject was funded by Centre for Regenerative Medicine andStem Cell Research at the Aga Khan University (AKU) andphilanthropic contributors.frontiersin.orgHassan and Rajput 10.3389/frhem.2024.1373408Conflict of interestThe authors declare that the research was conducted in theabsence of any commercial or financial relationships that could beconstrued as a potential conflict of interest.Publisher’s noteAll claims expressed in this article are solely those of the authorsand do not necessarily represent those of their affiliatedFrontiers in Hematology 10organizations, or those of the publisher, the editors and thereviewers. Any product that may be evaluated in this article, orclaim that may be made by its manufacturer, is not guaranteed orendorsed by the publisher.Supplementary materialThe Supplementary Material for this article can be found onlineat: https://www.frontiersin.org/articles/10.3389/frhem.2024.1373408/full#supplementary-materialReferences1. 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