RESUMO
Human pluripotent stem cells (hPSCs) can in theory give rise to any hematopoietic lineages, thereby offering opportunities for disease modeling, drug screening and cell therapies. However, gaps in our knowledge of the signaling requirements for the specification of human hematopoietic stem/progenitor cells (HSPCs), which lie at the apex of all hematopoietic lineages, greatly limit the potential of hPSC in hematological research and application. Transcriptomic analysis reveals aberrant regulation of WNT signaling during maturation of hPSC-derived hematopoietic progenitor cells (hPSC-HPCs), which results in higher mitochondria activity, misregulation of HOX genes, loss of self-renewal and precocious differentiation. These defects are partly due to the activation of the WNT target gene CDX2. Late-stage WNT inhibition improves the yield, self-renewal, multilineage differentiation, and transcriptional and metabolic profiles of hPSC-HPCs. Genome-wide mapping of transcription factor (TF) accessible chromatin reveals a significant overrepresentation of myeloid TF binding motifs in hPSC-HPCs, which could underlie their myeloid-biased lineage potential. Together our findings uncover a previously unappreciated dynamic requirement of the WNT signaling pathway during the specification of human HSPCs. Modulating the WNT pathway with small molecules normalizes the molecular differences between hPSC-HPCs and endogenous hematopoietic stem cells (HSCs), thereby representing a promising approach to improve the differentiation and function of hPSC-HPCs.
Assuntos
Diferenciação Celular , Células-Tronco Hematopoéticas , Células-Tronco Pluripotentes , Via de Sinalização Wnt , Humanos , Via de Sinalização Wnt/fisiologia , Diferenciação Celular/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Linhagem da Célula , Fator de Transcrição CDX2/metabolismo , Fator de Transcrição CDX2/genética , Perfilamento da Expressão Gênica , Fatores de Transcrição/metabolismoRESUMO
Hematopoietic stem cells (HSCs) are the rare cells responsible for the lifelong curative effects of hematopoietic cell (HC) transplantation. The demand for clinical-grade HSCs has increased significantly in recent decades, leading to major difficulties in treating patients. A promising but not yet achieved goal is the generation of HSCs from pluripotent stem cells. Here, we have obtained vector- and stroma-free transplantable HSCs by differentiating human induced pluripotent stem cells (hiPSCs) using an original one-step culture system. After injection into immunocompromised mice, cells derived from hiPSCs settle in the bone marrow and form a robust multilineage hematopoietic population that can be serially transplanted. Single-cell RNA sequencing shows that this repopulating activity is due to a hematopoietic population that is transcriptionally similar to human embryonic aorta-derived HSCs. Overall, our results demonstrate the generation of HSCs from hiPSCs and will help identify key regulators of HSC production during human ontogeny.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Humanos , Camundongos , Animais , Diferenciação Celular , Células-Tronco Hematopoéticas , Medula ÓsseaRESUMO
We previously demonstrated that intracardiac delivery of autologous peripheral blood-derived CD34+ stem cells (SCs), mobilized by granulocyte-colony stimulating factor (G-CSF) and collected by leukapheresis after myocardial infarction, structurally and functionally repaired the damaged myocardial area. When used for cardiac indication, CD34+ cells are now considered as Advanced Therapy Medicinal Products (ATMPs). We have industrialized their production by developing an automated device for ex vivo CD34+ -SC expansion, starting from a whole blood (WB) sample. Blood samples were collected from healthy donors after G-CSF mobilization. Manufacturing procedures included: (a) isolation of total nuclear cells, (b) CD34+ immunoselection, (c) expansion and cell culture recovery in the device, and (d) expanded CD34+ cell immunoselection and formulation. The assessment of CD34+ cell counts, viability, and immunophenotype and sterility tests were performed as quality tests. We established graft acceptance criteria and performed validation processes in three cell therapy centers. 59.4 × 106 ± 36.8 × 106 viable CD34+ cells were reproducibly generated as the final product from 220 ml WB containing 17.1 × 106 ± 8.1 × 106 viable CD34+ cells. CD34+ identity, genetic stability, and telomere length were consistent with those of basal CD34+ cells. Gram staining and mycoplasma and endotoxin analyses were negative in all cases. We confirmed the therapeutic efficacy of both CD34+ -cell categories in experimental acute myocardial infarct (AMI) in immunodeficient rats during preclinical studies. This reproducible, automated, and standardized expansion process produces high numbers of CD34+ cells corresponding to the approved ATMP and paves the way for a phase I/IIb study in AMI, which is currently recruiting patients. Stem Cells Translational Medicine 2019;8:822&832.
Assuntos
Antígenos CD34/genética , Automação Laboratorial/métodos , Citometria de Fluxo/métodos , Infarto do Miocárdio/terapia , Transplante de Células-Tronco de Sangue Periférico/métodos , Células-Tronco de Sangue Periférico/citologia , Adulto , Animais , Antígenos CD34/metabolismo , Células Cultivadas , Ensaios Clínicos como Assunto , Humanos , Imunofenotipagem/métodos , Masculino , Pessoa de Meia-Idade , Células-Tronco de Sangue Periférico/metabolismo , Cultura Primária de Células/métodos , RatosRESUMO
We here determine the influence of mesenchymal stem cell (MSC) therapy on the progression of solid tumors. The influence of MSCs was investigated in human colorectal cancer cells as well as in an immunocompetent rat model of colorectal carcinogenesis representative of the human pathology. Treatment with bone marrow (BM)-derived MSCs significantly reduced both cancer initiation and cancer progression by increasing the number of tumor-free animals as well as decreasing the number and the size of the tumors by half, thereby extending their lifespan. The attenuation of cancer progression was mediated by the capacity of the MSCs to modulate the immune component. Specifically, in the adenocarcinomas (ADKs) of MSC-treated rats, the infiltration of CD68+ monocytes/macrophages was 50% less while the presence of CD3+ lymphocytes increased almost twofold. The MSCs reprogrammed the macrophages to become regulatory cells involved in phagocytosis thereby inhibiting the production of proinflammatory cytokines. Furthermore, the MSCs decreased NK (Natural Killer) and rTh17 cell activities, Treg recruitment, the presence of CD8+ lymphocytes and endothelial cells while restoring Th17 cell activity. The expression of miR-150 and miR-7 increased up to fivefold indicating a likely role for these miRNAs in the modulation of tumor growth. Importantly, MSC administration limited the damage of healthy tissues and attenuated tumor growth following radiotherapy. Taken together, we here show that that MSCs have durable action on colon cancer development by modulating the immune component of the tumor microenvironment. In addition, we identify two miRNAs associated with the capacity of MSCs to attenuate cancer growth. Stem Cells Translational Medicine 2019;8:285&300.
Assuntos
Neoplasias Colorretais/imunologia , Neoplasias Colorretais/terapia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia , Microambiente Tumoral/fisiologia , Animais , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Técnicas de Cocultura/métodos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Células Endoteliais/citologia , Células Endoteliais/imunologia , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Ratos , Ratos Sprague-Dawley , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Células Th17/metabolismo , Microambiente Tumoral/imunologiaRESUMO
The genetic landscape of adult acute myeloid leukemias (AML) has been recently unraveled. However, due to their genetic heterogeneity, only a handful of markers are currently used for the evaluation of minimal residual disease (MRD). Recent studies using multi-target strategies indicate that detection of residual mutations in less than 5% of cells in complete remission is associated with a better survival. Here, in a series of 69 AMLs with known clonal architecture, we design a clone-specific strategy based on fluorescent in situ hybridization and high-sensitivity next generation sequencing to detect chromosomal aberrations and mutations, respectively, in follow-up samples. The combination of these techniques allows tracking chromosomal and genomic lesions down to 0.5-0.4% of the cell population in remission samples. By testing all lesions in follow-up samples from 65 of 69 evaluable patients, we find that initiating events often persist and appear to be, on their own, inappropriate markers to predict short-term relapse. In contrast, the persistence of two or more lesions in more than 0.4% of the cells from remission samples is strongly associated with lower leukemia-free and overall survivals in univariate and multivariate analyses. Although larger prospective studies are needed to extend these results, our data show that a personalized, clone-specific, MRD follow up strategy is feasible in the vast majority of AML cases.
Assuntos
Evolução Clonal/genética , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Biomarcadores Tumorais , Aberrações Cromossômicas , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Mutação , Medicina de Precisão , Prognóstico , Adulto JovemRESUMO
In acute myeloid leukaemia (AML) initiating pre-leukaemic lesions can be identified through three major hallmarks: their early occurrence in the clone, their persistence at relapse and their ability to initiate multilineage haematopoietic repopulation and leukaemia in vivo. Here we analyse the clonal composition of a series of AML through these characteristics. We find that not only DNMT3A mutations, but also TET2, ASXL1 mutations, core-binding factor and MLL translocations, as well as del(20q) mostly fulfil these criteria. When not eradicated by AML treatments, pre-leukaemic cells with these lesions can re-initiate the leukaemic process at various stages until relapse, with a time-dependent increase in clonal variegation. Based on the nature, order and association of lesions, we delineate recurrent genetic hierarchies of AML. Our data indicate that first lesions, variegation and treatment selection pressure govern the expansion and adaptive behaviour of the malignant clone, shaping AML in a time-dependent manner.
Assuntos
Evolução Clonal/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Animais , Sequência de Bases , Células Clonais , Epigênese Genética , Rearranjo Gênico/genética , Hematopoese , Humanos , Camundongos , Mutação/genética , Análise de Célula Única , Fatores de TempoRESUMO
There is little information on the fate of infused mesenchymal stem cells (MSCs) and long-term side effects after irradiation exposure. We addressed these questions using human MSCs (hMSCs) intravenously infused to nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice submitted to total body irradiation (TBI) or local irradiation (abdominal or leg irradiation). The animals were sacrificed 3 to 120 days after irradiation and the quantitative and spatial distribution of hMSCs were studied by polymerase chain reaction (PCR). Following their infusion into nonirradiated animals, hMSCs homed to various tissues. Engraftment depended on the dose of irradiation and the area exposed. Total body irradiation induced an increased hMSC engraftment level compared to nonirradiated mice, while local irradiations increased hMSC engraftment locally in the area of irradiation. Long-term engraftment of systemically administered hMSCs in NOD/SCID mice increased significantly in response to tissue injuries produced by local or total body irradiation until 2 weeks then slowly decreased depending on organs and the configuration of irradiation. In all cases, no tissue abnormality or abnormal hMSCs proliferation was observed at 120 days after irradiation. This work supports the safe and efficient use of MSCs by injection as an alternative approach in the short- and long-term treatment of severe complications after radiotherapy for patients refractory to conventional treatments.
RESUMO
Blood-transfusion centers regularly face the challenge of donor blood shortages, especially for rare blood groups. The possibility of producing universal red blood cells from stem cells industrially has become a possible alternative since the successful injection of blood generated in vitro into a human being in 2011. Although there remains many biological and regulatory issues concerning the efficacy and safety of this new product, the major challenge today for future clinical applications is switching from the current limited 2-dimensional production techniques to large-scale 3-dimensional bioreactors. In addition to requiring technological breakthroughs, the whole process also has to become at least five-fold more cost-efficient to match the current prices of high-quality blood products. The current review sums up the main biological advances of the past decade, outlines the key biotechnological challenges for the large-scale cost-effective production of red blood cells, proposes solutions based on strategies used in the bioindustry and presents the state-of-the-art of large-scale blood production.
Assuntos
Técnicas de Cultura de Células/economia , Técnicas de Cultura de Células/métodos , Eritrócitos , Células-Tronco Hematopoéticas , Animais , Reatores Biológicos , Transfusão de Sangue , Eritrócitos/citologia , Células-Tronco Hematopoéticas/citologia , HumanosRESUMO
To evaluate the potential therapeutic effect of the infusion of hMSCs for the correction of liver injuries, we performed total body radiation exposure of NOD/SCID mice. After irradiation, mir-27b level decreases in liver, increasing the directional migration of hMSCs by upregulating SDF1 α . A significant increase in plasmatic transaminases levels, apoptosis process in the liver vascular system, and in oxidative stress were observed. hMSC injection induced a decrease in transaminases levels and oxidative stress, a disappearance of apoptotic cells, and an increase in Nrf2, SOD gene expression, which might reduce ROS production in the injured liver. Engrafted hMSCs expressed cytokeratin CK18 and CK19 and AFP genes indicating possible hepatocyte differentiation. The presence of hMSCs expressing VEGF and Ang-1 in the perivascular region, associated with an increased expression of VEGFr1, r2 in the liver, can confer a role of secreting cells to hMSCs in order to maintain the endothelial function. To explain the benefits to the liver of hMSC engraftment, we find that hMSCs secreted NGF, HGF, and anti-inflammatory molecules IL-10, IL1-RA contributing to prevention of apoptosis, increasing cell proliferation in the liver which might correct liver dysfunction. MSCs are potent candidates to repair and protect healthy tissues against radiation damages.
Assuntos
Diferenciação Celular , Hepatócitos/citologia , Fígado/lesões , Células-Tronco Mesenquimais/citologia , Animais , Antioxidantes/metabolismo , Quimiocina CXCL12/metabolismo , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Fígado/efeitos da radiação , Células-Tronco Mesenquimais/metabolismo , Camundongos , MicroRNAs/metabolismo , Irradiação Corporal TotalRESUMO
Radiotherapy may induce irreversible damage on healthy tissues surrounding the tumor. It has been reported that the majority of patients receiving pelvic radiation therapy show early or late tissue reactions of graded severity as radiotherapy affects not only the targeted tumor cells but also the surrounding healthy tissues. The late adverse effects of pelvic radiotherapy concern 5% to 10% of them, which could be life threatening. However, a clear medical consensus concerning the clinical management of such healthy tissue sequelae does not exist. Although no pharmacologic interventions have yet been proven to efficiently mitigate radiotherapy severe side effects, few preclinical researches show the potential of combined and sequential pharmacological treatments to prevent the onset of tissue damage. Our group has demonstrated in preclinical animal models that systemic mesenchymal stromal cell (MSC) injection is a promising approach for the medical management of gastrointestinal disorder after irradiation. We have shown that MSCs migrate to damaged tissues and restore gut functions after irradiation. We carefully studied side effects of stem cell injection for further application in patients. We have shown that clinical status of four patients suffering from severe pelvic side effects resulting from an over-dosage was improved following MSC injection in a compationnal situation.
RESUMO
High dose radiation exposures involving medical treatments or accidental irradiation may lead to extended damage to the irradiated tissue. Alleviation or even eradication of irradiation induced adverse events is therefore crucial. Because developments in cell therapy have brought some hope for the treatment of tissues damages induced by irradiation, the Institute for Radiation and Nuclear Safety contributed to establish the clinical guidelines for the management of accidentally irradiated victims and to provide the best supportive care to patients all over the world. In the past 15 years, we contributed to develop and test cell therapy for protection against radiation side effects in several animal models, and we proposed mechanisms to explain the benefit brought by this new therapeutic approach. We established the proof of concept that mesenchymal stem cells (MSCs) migrate to damaged tissues in the nonobese diabetic/severe combined immunodeficiency immunotolerant mice model and in non-human primate after radiation exposure. We showed that the intravenous injection of MSCs sustains hematopoiesis after total body irradiation, improves wound healing after radiodermatitis and protects gut function from irradiation damages. Thanks to a tight collaboration with clinicians from several French hospitals, we report successful treatments of therapeutic/accidental radiation damages in several victims with MSC infusions for hematopoiesis correction, radio-induced burns, gastrointestinal disorders and protection homeostatic functions of gut management after radio-therapy.
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The generation in vitro of cultured red blood cells (cRBC) could become an alternative to classical transfusion products. However, even when derived from healthy donors, the cRBC generated in vitro from hematopoietic stem cells may display alterations resulting from a poor controlled production process. In this context, we attempted to monitor the quality of the transfusion products arising from new biotechnologies. For that purpose, we developed an in vitro erythrophagocytosis (EP) test with the murine fibroblast cell line MS-5 and human macrophages (reference method). We evaluated 38 batches of cRBC, at the stage of reticulocyte, generated from CD34(+) cells isolated from placental blood or by leukapheresis. We showed that (i) the EP test performed with the MS-5 cell line was sensitive and can replace human macrophages for the evaluation of cultured cells. (ii) The EP tests revealed disparities among the batches of cRBC. (iii) The viability of the cells (determined by calcein-AM test), the expression of CD47 (antiphagocytosis receptor) and the externalization of phosphatidylserine (PS, marker of phagocytosis) were not critical parameters for the validation of the cRBC. (iv) Conversely, the cell deformability determined by ektacytometry was inversely correlated with the intensity of the phagocytic index. Assuming that the culture conditions directly influence the quality of the cell products generated, optimization of the production mode could benefit from the erythrophagocytosis test.
Assuntos
Eritrócitos/citologia , Animais , Antígeno CD47/análise , Células Cultivadas/química , Células Cultivadas/citologia , Células Cultivadas/fisiologia , Envelhecimento Eritrocítico , Deformação Eritrocítica , Membrana Eritrocítica/química , Transfusão de Eritrócitos , Eritrócitos/química , Eritropoese , Fibroblastos/fisiologia , Fluoresceínas/análise , Corantes Fluorescentes/análise , Células-Tronco Hematopoéticas/citologia , Humanos , Leucaférese , Macrófagos/fisiologia , Lipídeos de Membrana/análise , Camundongos , Fagocitose , Fosfatidilserinas/análise , Reticulócitos/citologiaRESUMO
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 this in vitro model, maturation that is enucleation reaches a lower level compared to adult sources as observed for embryonic or iP, but, interestingly, they (i) displayed a dramatic in vitro expansion (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 for ex vivo RBCS 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.
RESUMO
In vitro RBC production from stem cells could represent an alternative to classic transfusion products. Until now the clinical feasibility of this concept has not been demonstrated. We addressed the question of the capacity of cultured RBCs (cRBCs) to survive in humans. By using a culture protocol permitting erythroid differentiation from peripheral CD34(+) HSC, we generated a homogeneous population of cRBC functional in terms of their deformability, enzyme content, capacity of their hemoglobin to fix/release oxygen, and expression of blood group antigens. We then demonstrated in the nonobese diabetes/severe combined immunodeficiency mouse that cRBC encountered in vivo the conditions necessary for their complete maturation. These data provided the rationale for injecting into one human a homogeneous sample of 10(10) cRBCs generated under good manufacturing practice conditions and labeled with (51)Cr. The level of these cells in the circulation 26 days after injection was between 41% and 63%, which compares favorably with the reported half-life of 28 ± 2 days for native RBCs. Their survival in vivo testifies globally to their quality and functionality. These data establish the proof of principle for transfusion of in vitro-generated RBCs and path the way toward new developments in transfusion medicine. This study is registered at http://www.clinicaltrials.gov as NCT0929266.
Assuntos
Transfusão de Eritrócitos/métodos , Animais , Antígenos CD34/sangue , Antígenos de Grupos Sanguíneos/sangue , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Envelhecimento Eritrocítico , Deformação Eritrocítica , Eritrócitos/citologia , Eritrócitos/imunologia , Eritrócitos/metabolismo , Eritropoese , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Hemoglobinas/metabolismo , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante HeterólogoRESUMO
PURPOSE OF REVIEW: In the context of chronic blood supply difficulties, generating cultured red blood cells (cRBCs) in vitro after amplification of stem cells makes sense. This review will focus on the recent findings about the generation of erythroid cells from induced pluripotent stem (iPS) cells and deals with the hurdles and next developments that will occur. RECENT FINDINGS: The most proliferative source of stem cells for generating cRBCs is the cord blood, but this source is limited in terms of hematopoietic stem cells and dependent on donations. Pluripotent stem cells are thus the best candidates and potential sources of cRBCs. Critical advances have led towards the in-vitro production of functional RBCs from iPS cells in the last few years. SUMMARY: Because iPS cells can proliferate indefinitely and can be selected for a phenotype of interest, they are potential candidates to organize complementary sources of RBCs for transfusion. Proof of concept of generating cRBCs from iPS cells has been performed, but the procedures need to be optimized to lead to clinical application in blood transfusion. Several crucial points remain to be resolved. Notably these include the choice of the initial cell type to generate iPS cells, the method of reprogramming, that is, to ensure the safety of iPS cells as clinical grade, the optimization of erythrocyte differentiation, and the definition of good manufacturing practice (GMP) conditions for industrial production.
Assuntos
Eritrócitos/citologia , Células-Tronco Pluripotentes/citologia , Animais , Transfusão de Sangue/métodos , Humanos , CamundongosRESUMO
The transfusion of red blood cells (RBCs) is now considered a well-settled and essential therapy. However, some difficulties and constraints still occur, such as long-term blood product shortage, blood donor population aging, known and yet unknown transfusion-transmitted infectious agents, growing cost of the transfusion supply chain management, and the inescapable blood group polymorphism barrier. Red blood cells can be now cultured in vitro from human hematopoietic, human embryonic, or human-induced pluripotent stem cells (hiPSCs). The highly promising hiPSC technology represents a potentially unlimited source of RBCs and opens the door to the revolutionary development of a new generation of allogeneic transfusion products. Assuming that in vitro large-scale cultured RBC production efficiently operates in the near future, we draw here some futuristic but realistic scenarios regarding potential applications for alloimmunized patients and those with a rare blood group. We retrospectively studied a cohort of 16,486 consecutive alloimmunized patients (10-year period), showing 1 to 7 alloantibodies with 361 different antibody combinations. We showed that only 3 hiPSC clones would be sufficient to match more than 99% of the 16,486 patients in need of RBC transfusions. The study of the French National Registry of People with a Rare Blood Phenotype/Genotype (10-year period) shows that 15 hiPSC clones would cover 100% of the needs in patients of white ancestry. In addition, one single hiPSC clone would meet 73% of the needs in alloimmunized patients with sickle cell disease for whom rare cryopreserved RBC units were required. As a result, we consider that a very limited number of RBC clones would be able to not only provide for the need for most alloimmunized patients and those with a rare blood group but also efficiently allow for a policy for alloimmunization prevention in multiply transfused patients.
Assuntos
Células-Tronco Adultas , Armazenamento de Sangue/métodos , Tipagem e Reações Cruzadas Sanguíneas/métodos , Eritrócitos/fisiologia , Isoanticorpos/imunologia , Células-Tronco Pluripotentes , Adulto , Animais , Coleta de Amostras Sanguíneas/métodos , Diferenciação Celular , Transfusão de Eritrócitos/métodos , Humanos , Isoanticorpos/efeitos adversosRESUMO
This article describes the research in progress that will permit the large-scale production of human red blood cells from hematopoietic stem cells. It also discusses the current state of this research, suggests the obstacles to be overcome to pass from the laboratory model to clinical practice, and analyzes the possible indications in the medium and long term. The potential interest of pluripotent stem cells as an unlimited source of red blood cells is considered. If it succeeds, this new approach could mark a considerable advance in the field of transfusion.
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Substitutos Sanguíneos , Transfusão de Eritrócitos/métodos , Eritrócitos/citologia , Células-Tronco Hematopoéticas/citologia , Adulto , Antígenos de Grupos Sanguíneos/biossíntese , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Hemoglobinas/química , Humanos , Marketing de Serviços de Saúde , Reticulócitos/citologiaRESUMO
BACKGROUND: Ex vivo manufacture of red blood cells from stem cells is a potential means to ensure an adequate and safe supply of blood cell products. Advances in somatic cell reprogramming of human induced pluripotent stem cells have opened the door to generating specific cells for cell therapy. Human induced pluripotent stem cells represent a potentially unlimited source of stem cells for erythroid generation for transfusion medicine. DESIGN AND METHODS: We characterized the erythroid differentiation and maturation of human induced pluripotent stem cell lines obtained from human fetal (IMR90) and adult fibroblasts (FD-136) compared to those of a human embryonic stem cell line (H1). Our protocol comprises two steps: (i) differentiation of human induced pluripotent stem cells by formation of embryoid bodies with indispensable conditioning in the presence of cytokines and human plasma to obtain early erythroid commitment, and (ii) differentiation/maturation to the stage of cultured red blood cells in the presence of cytokines. The protocol dispenses with major constraints such as an obligatory passage through a hematopoietic progenitor, co-culture on a cellular stroma and use of proteins of animal origin. RESULTS: We report for the first time the complete differentiation of human induced pluripotent stem cells into definitive erythrocytes capable of maturation up to enucleated red blood cells containing fetal hemoglobin in a functional tetrameric form. CONCLUSIONS: Red blood cells generated from human induced pluripotent stem cells pave the way for future development of allogeneic transfusion products. This could be done by banking a very limited number of red cell phenotype combinations enabling the safe transfusion of a great number of immunized patients.
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Eritrócitos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Linhagem Celular , Citocinas/farmacologia , Transfusão de Eritrócitos , HumanosRESUMO
BACKGROUND: Anemia is a characteristic of myelodysplastic syndromes, such as the rare 5q- syndrome, but its mechanism remains unclear. In particular, data are lacking on the terminal phase of differentiation of erythroid cells (enucleation) in myelodysplastic syndromes. DESIGN AND METHODS: We used a previously published culture model to generate mature red blood cells in vitro from human hematopoietic progenitor cells in order to study the pathophysiology of the 5q- syndrome. Our model enables analysis of cell proliferation and differentiation at a single cell level and determination of the enucleation capacity of erythroid precursors. RESULTS: The erythroid commitment of 5q(del) clones was not altered and their terminal differentiation capacity was preserved since they achieved final erythroid maturation (enucleation stage). The drop in red blood cell production was secondary to the decrease in the erythroid progenitor cell pool and to impaired proliferative capacity. RPS14 gene haploinsufficiency was related to defective erythroid proliferation but not to differentiation capacity. CONCLUSIONS: The 5q- syndrome should be considered a quantitative rather than qualitative bone marrow defect. This observation might open the way to new therapeutic concepts.