scholarly journals The Future of Red Cell Transfusion Lies in Cultured Red Cells

2021 ◽  
Author(s):  
Rizwan Javed ◽  
Lorraine Flores ◽  
Saurabh Jayant Bhave ◽  
Asheer Jawed ◽  
Deepak Kumar Mishra
Keyword(s):  
Stem Cells ◽  
Large Scale ◽  
Ex Vivo ◽  
Blood Groups ◽  
Red Cells ◽  
Controlled Clinical Trial ◽  
Scale Production ◽  
Red Cell ◽  
Potential Cost ◽  
Hematopoietic Stem

AbstractBlood is a very important resource for healthcare-based services and there has been a consistently increasing demand for it in most parts of the world. Poor volunteer-based collection system, high-risk of transfusion-transmitted infections, and emergence of new pathogens as evident from the ongoing Coronavirus Disease 2019 (COVID-19) pandemic are potential challenges to the global healthcare systems. It is imperative to explore safe and reliable alternatives to red cell transfusions. Ex vivo culture of red cells (cRBCs) from different sources such as hematopoietic stem cells (HSCs), pluripotent stem cells, and immortalized progenitors (e.g., BELA-2 cells) could revolutionize transfusion medicine. cRBC could be of great diagnostic and therapeutic utility. It may provide a backup in times of acute shortages in patients with rare blood groups, and in cases with multiple antibodies or sickle cell anemia. The CRISP-Cas9 system has been used to develop personalized, multi-compatible RBCs for diagnostic reagents and patients with multiple allo-antibodies. cRBC could be practically feasible for pediatric patients, who require small quantities of red cell transfusions. cRBC produced under good manufacturing practice (GMP) conditions has been reported to survive in human blood circulation for more than 26 days. Recently, a phase I randomized controlled clinical trial called RESTORE was initiated to assess the survival and recovery of cRBCs. However, feasible technological advancement is required to produce enough cRBCs for clinical use. It is crucial to identify sustainable sources for large-scale production of clinically useful cRBCs. Although the potential cost of one unit of cRBC is extrapolated to be around US$ 8000, it is a life-saving product for patients having rare blood groups and is a “ready to use” source of phenotype-matched, homogenous young red cells in emergency situations.

Large-scale production and directed induction of functional dendritic cells ex vivo from serum-free expanded human hematopoietic stem cells

Cytotherapy ◽  
2019 ◽  
Vol 21 (7) ◽  
pp. 755-768 ◽  
Author(s):  
Shu-Ching Hsu ◽  
Li-Cheng Lu ◽  
Kuang-Yu Chan ◽  
Chien-Hsun Huang ◽  
Shih-Lung Cheng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dendritic Cells ◽  
Hematopoietic Stem Cells ◽  
Large Scale ◽  
Ex Vivo ◽  
Scale Production ◽  
Hematopoietic Stem ◽  
Serum Free ◽  
Large Scale Production

Non-Random Lentiviral Vector Insertions in Bone Marrow Progenitors from Fanconi Anemia Patients

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2358-2358
Author(s):  
Ali Nowrouzi ◽  
Africa Gonzales-Murillo ◽  
Anna Paruzynski ◽  
Ariana Jacome ◽  
Paula Rio ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Fanconi Anemia ◽  
Large Scale ◽  
Random Distribution ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
In Cis

Abstract Improved protocols using lentiviral vectors have been established with minimal cytokine exposure and short transduction times proving more suitable for overcoming the disease-specific challenge in correcting functionally defective hematopoietic stem cells (HSCs) of Fanconi Anemia (FA) patients. Bone marrow (BM) cells from FA patients were transduced ex vivo with lentiviral vectors (LVs) expressing FANCA and/or EGFP using optimized conditions to preserve the repopulating properties of the primitive hematopoietic stem cells (manuscript submitted). In a forward preclinical screening of possible LV-induced side effects we analyzed the insertional inventory in colonies generated by FA BM cells previously transduced with the LVs. We have established and optimized DNA and RNA isolation procedures for minimal cell numbers, suitable for large scale screening of colony forming cell (CFC) derived colonies by linear amplification-mediated PCR (LAM-PCR) and massive parallel pyrosequencing (454 GS Flx system; Roche). This approach is applicable for detecting early indicators of clonal selection, and is based on the analysis of common integration sites (CIS) and non-random distribution of vector insertions in particular genomic loci. From a total of 180 CFC-derived colonies expressing the EGFP LV marker gene, 298 vector insertions could be sequenced and mapped to the human genome. The analysis of vector targeted gene coding regions showed a non-random genomic distribution of LV insertions, with a significant overrepresentation of RefSeq genes that are part of distinct functional categories. Accordingly vector associated genes are predominantly involved in cellular signal cascades regulated by the MAP Kinase family known to be involved in a wide variety of cellular processes such as proliferation, differentiation, transcription regulation and development. Apart from the observed high integration frequency in genes (>80%), partial loss of vector LTR nucleotides was detected in >10% of the integrants (3–25bp). Notably, >20% of the lentiviral insertions were found to be located in CIS of predominantly 2nd order. Further screening assays of LV transduced CFC-derived colonies will allow a deeper investigation in the functional consequences of such CIS targeting in gene therapy protocols of FA. However our results suggest that the LV transduction of FA BM progenitors leads to a relatively high frequency of insertions in CIS which may be indicative of an insertion based (specific) selection mechanism. We herby show that the ex vivo large scale integration site analyses of CFC-derived colonies from patients considered to undergo gene therapeutic treatments constitutes a robust approach, which combined with mouse preclinical biosafety studies will help to improve the safety of clinical gene therapy protocols. The non-random distribution of LV integrations in CIS associated genes and in genes involved in particular cellular pathways may be indicative for the altered biochemical pathways characteristic of FA stem cells, with reported defects in DNA repair and self-renewal.

scholarly journals Human Fetal Liver: AnIn VitroModel of Erythropoiesis

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Guillaume Pourcher ◽  
Christelle Mazurier ◽  
Yé Yong King ◽  
Marie-Catherine Giarratana ◽  
Ladan Kobari ◽  
...  
Keyword(s):  
Stem Cells ◽  
Large Scale ◽  
Adult Stem Cells ◽  
Fetal Liver ◽  
Es Cells ◽  
Embryonic Stem ◽  
Scale Production ◽  
Cloning Efficiency ◽  
Hematopoietic Stem

We previously described the large-scale production of RBCs from hematopoietic stem cells (HSCs) of diverse sources. Our present efforts are focused to produce RBCs thanks to an unlimited source of stem cells. Human embryonic stem (ES) cells or induced pluripotent stem cell (iPS) are the natural candidates. Even if the proof of RBCs production from these sources has been done, their amplification ability is to date not sufficient for a transfusion application. In this work, our protocol of RBC production was applied to HSC isolated from fetal liver (FL) as an intermediate source between embryonic and adult stem cells. We studied the erythroid potential of FL-derived CD34+cells. In thisin vitromodel, maturation that is enucleation reaches a lower level compared to adult sources as observed for embryonic or iP, but, interestingly, they (i) displayed a dramaticin vitroexpansion (100-fold more when compared to CB CD34+) and (ii) 100% cloning efficiency in hematopoietic progenitor assays after 3 days of erythroid induction, as compared to 10–15% cloning efficiency for adult CD34+cells. This work supports the idea that FL remains a model of study and is not a candidate forex vivoRBCS production for blood transfusion as a direct source of stem cells but could be helpful to understand and enhance proliferation abilities for primitive cells such as ES cells or iPS.

Nanofiber-Based Expansion and Differentiation Technology for High-Volume Ex Vivo Production of Reticulocytes for P. Vivax Malaria Research

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2357-2357
Author(s):  
Hong Wang ◽  
Adam M Sorkin ◽  
Ramasamy Sakthivel
Keyword(s):  
Cord Blood ◽  
Large Scale ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Rapid Development ◽  
High Volume ◽  
In Vitro Models ◽  
Scale Production ◽  
Hematopoietic Stem

Abstract Abstract 2357 Infection by Plasmodium Vivax (P. Vivax) is the most common cause of Sleeping Malaria. P. Vivax and other plasmodia have grown increasingly resistant to antimalarial drugs. Introduced by mosquito bite, P. vivax sporozoites enter circulation and preferentially penetrate reticulocytes by attaching to the Fya and Fyb Duffy antigen/chemokine receptor (DARC) via PvRBP-1 and PvRBP-2 proteins located at their apical poles. Once in a reticulocyte, the parasite begins to reproduce asexually, releasing of thousands of merozoites into circulation. At this point, merozoites can also enter the liver and triggering relapses months or years later. The emergence of drug-resistant strains of p. vivax has stimulated development of new vaccines and treatments, but progress has been slowed by the dearth of reliable screening platforms. Many vaccine candidates have been developed to act upon vivax merozoites by preventing binding of PvRBP-1 and 2 to DARC, thereby arresting reproduction. However, there is a distinct lack of in vitro models to evaluate candidates that employ this mechanism. We are addressing this issue with a novel ex vivo expansion and differentiation technology for large-scale production of DARC expressing reticulocytes for in vitro P. vivax infection studies. This technology comprises an expansion system that can produce high yields of hematopoietic precursors (CD133+/CD34+ cells) from a variety of sources (marrow, peripheral blood, and cord blood), and a differentiation system to produce a relatively pure population of enucleated erythrocytes. In this study, we have refined the polyethersulfone (PES) nanofiber-based culturing system containing growth factors and cytokines in a serum-free media, to expand hematopoietic stem and progenitor cells (HSPC) ex vivo. This expansion technology allows rapid 200-fold ex vivo proliferation within 7 days of umbilical cord blood derived CD133+/CD34+ HSPCs from a DARC+ donor. Following expansion, over 50% of these cells retained HSPC phenotype (expression of CD34+). We have subsequently demonstrated that feeder layer free three-step differentiation of nanofiber-expanded cells using cytokines results in a population containing predominately enucleated reticulocyte-like cells. At 21 days of differentiation, cells had expanded 50-fold. Around 41% of cells were enucleated reticulocytes. These cells expressed glycophorin-A, a major sialoglycoprotein present on the human erythrocyte membrane. ∼28% of cells were CD36+, and ∼70% were CD71+ indicating an erythroid lineage. These results suggest that this technology can produce a population of DARC+ reticulocytes that is ∼5,000-fold greater than the starting population of HSPCs. We are partnering with leading malaria vaccine researchers to demonstrate that these reticulocytes can be parasitized by p. vivax. We believe that this will provide a unique platform to jumpstart research of malaria parasites and enable rapid development of effective vaccines. Further development of this technology may also have significant implications for large-scale ex vivo production of erythrocytes for general use. Reticulocyte-like cells and expelled nuclei during differentiation of nanofiber-expanded HSPC. Disclosures: No relevant conflicts of interest to declare.

Expansion of Red Blood Cells for Transfusion

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. SCI-46-SCI-46
Author(s):  
Anna Rita F Migliaccio ◽  
Carolyn Whitsett ◽  
Giovanni Migliaccio
Keyword(s):  
Stem Cells ◽  
Blood Transfusion ◽  
Blood Supply ◽  
Ex Vivo ◽  
Red Cells ◽  
Blood Group Antigens ◽  
Red Cell ◽  
Erythroid Cells ◽  
Red Cell Transfusion ◽  
Safety Considerations

Abstract Abstract SCI-46 Blood transfusion, the earliest form of cell replacement therapy, has become indispensable for modern medicine making the safety and adequacy of the blood supply a national priority. The US blood supply is adequate overall because in 2006 the number of blood units collected exceed by 7.8% the number of those transfused. However, issues surrounding blood transfusion, such as sporadic shortages and potential adverse events to recipients (related to changes in red cell physiology during storage and alloimmunization in chronically transfused patients) prompted past and current efforts to develop alternative transfusion products. Recently, the culture conditions to generate erythroid cells have greatly improved making the production of a transfusion product ex-vivo a theoretically possible, although expensive, proposition. This recognition is inspiring several investigators to develop production processes for ex-vivo generation of red cell transfusion products. A proof-of-concept demonstrating that ex-vivo generated red cells protect mice from experimentally induced lethal anemia has been obtained. Alternative sources of stem cells which include human embryonic stem cells (hESC) and induced pluripotency stem cells (iPS), are being explored. Since red cells do not have a nucleus, safety considerations suggest that they may represent the first cell therapy product to be generated from hESC and iPS. In addition, discarded hematopoietic stem cells present in adult and cord blood donations may theoretically generate numbers of red cells ex-vivo sufficient for transfusion. Affordable clinical grade humanized culture media have also been developed. Possible differences in immunological and biological properties of erythroid cells from different sources are under investigation. These differences include size, levels of activity of glycolytic enzymes and carbonic anhydrase, expression of different isozymes, hemoglobin and antigenic profiles (HLA class II antigens). This last aspect is particularly important because ex-vivo expanded red cells pose the same risk for infection and incompatibility as any transfusion product but pose unique antigenic risks. Since expression of blood group antigens is susceptible to post-transcriptional modifications, the ex-vivo expansion process itself may induce antigenic variability. Therefore, even cells generated from completely matched stem cell sources may induce auto-immunity and/or appear incompatible. Regarding the identity of ex-vivo generated red cell transfusion products, a conservative approach would be to define them as “enucleated red cells”. In principle, however, ex-vivo generated erythroblasts may also serve as transfusion product. Since they undergo 4–64 further divisions and reduce iron overload, they may represent a more potent transfusion product for patients that require chronic transfusion. The clinical use of these cells, however, may involve development of specific procedures to facilitate their homing/maturation in the erythroid niches of the recipients. In summary, on the basis of these cost, logistic and safety considerations we hypothesize that the clinical application of ex-vivo expanded erythroblasts will involve in sequence, drug discovery for personalized therapy, systemic drug delivery, genotypically matched transfusion for alloimmunized patients and then transfusion in the general population. Disclosures: No relevant conflicts of interest to declare.

In vitro large scale production of megakaryocytes to functional platelets from human hematopoietic stem cells

2018 ◽  
Vol 505 (1) ◽  
pp. 168-175 ◽  
Author(s):  
Pasupuleti Santhosh Kumar ◽  
Chodimella Chandrasekhar ◽  
Lokanathan Srikanth ◽  
Potukuchi Venkata Gurunadha Krishna Sarma
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Large Scale ◽  
Scale Production ◽  
Hematopoietic Stem ◽  
Large Scale Production

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

2020 ◽  
Vol 15 ◽  
Author(s):  
Fatima Aerts-Kaya
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stress Conditions ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

A Review of Evaluating Hematopoietic Stem Cells Derived from Umbilical Cord Blood's Expansion and Homing

2020 ◽  
Vol 15 (3) ◽  
pp. 250-262
Author(s):  
Maryam Islami ◽  
Fatemeh Soleimanifar
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Umbilical Cord ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Hematologic Malignancies ◽  
Future Directions ◽  
Hematopoietic Stem ◽  
Efficient Procedure ◽  
Hematopoietic Recovery

Transplantation of hematopoietic stem cells (HSCs) derived from umbilical cord blood (UCB) has been taken into account as a therapeutic approach in patients with hematologic malignancies. Unfortunately, there are limitations concerning HSC transplantation (HSCT), including (a) low contents of UCB-HSCs in a single unit of UCB and (b) defects in UCB-HSC homing to their niche. Therefore, delays are observed in hematopoietic and immunologic recovery and homing. Among numerous strategies proposed, ex vivo expansion of UCB-HSCs to enhance UCB-HSC dose without any differentiation into mature cells is known as an efficient procedure that is able to alter clinical treatments through adjusting transplantation-related results and making them available. Accordingly, culture type, cytokine combinations, O2 level, co-culture with mesenchymal stromal cells (MSCs), as well as gene manipulation of UCB-HSCs can have effects on their expansion and growth. Besides, defects in homing can be resolved by exposing UCB-HSCs to compounds aimed at improving homing. Fucosylation of HSCs before expansion, CXCR4-SDF-1 axis partnership and homing gene involvement are among strategies that all depend on efficiency, reasonable costs, and confirmation of clinical trials. In general, the present study reviewed factors improving the expansion and homing of UCB-HSCs aimed at advancing hematopoietic recovery and expansion in clinical applications and future directions.

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