Precision and prognostic value of clone-specific minimal residual disease in acute myeloid leukemia.

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.

Molecular signature of erythroblast enucleation in human embryonic stem cells.

While enucleation is a critical step in the terminal differentiation of human red blood cells, the molecular mechanisms underlying this unique process remain unclear. To investigate erythroblast enucleation, we studied the erythroid differentiation of human embryonic stem cells (hESCs), which provide a unique model for deeper understanding of the development and differentiation of multiple cell types. First, using a two-step protocol, we demonstrated that terminal erythroid differentiation from hESCs is directly dependent on the age of the embryoid bodies. Second, by choosing hESCs in two extreme conditions of erythroid culture, we obtained an original differentiation model which allows one to study the mechanisms underlying the enucleation of erythroid cells by analyzing the gene and miRNA (miR) expression profiles of cells from these two culture conditions. Third, using an integrated analysis of mRNA and miR expression profiles, we identified five miRs potentially involved in erythroblast enucleation. Finally, by selective knockdown of these five miRs we found miR-30a to be a regulator of erythroblast enucleation in hESCs.

Biological validation of bio-engineered red blood cell productions.

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.

Proof of principle for transfusion of in vitro-generated red blood cells.

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.

In vitro generated Rh(null) red cells recapitulate the in vivo deficiency: a model for rare blood group phenotypes and erythroid membrane disorders.

Lentiviral modification combined with ex vivo erythroid differentiation was used to stably inhibit RhAG expression, a critical component of the Rh(rhesus) membrane complex defective in the Rh(null) syndrome. The cultured red cells generated recapitulate the major alterations of native Rh(null) cells regarding antigen expression, membrane deformability, and gas transport function, providing the proof of principle for their use as model of Rh(null) syndrome and to investigate Rh complex biogenesis in human primary erythroid cells. Using this model, we were able to reveal for the first time that RhAG extinction alone is sufficient to explain ICAM-4 and CD47 loss observed on native Rh(null) RBCs. Together with the effects of RhAG forced expression in Rh(null) progenitors, this strongly strengthens the hypothesis that RhAG is critical to Rh complex formation. The strategy is also promising for diagnosis purpose in order to overcome the supply from rare blood donors and is applicable to other erythroid defects and rare phenotypes, providing models to dissect membrane biogenesis of multicomplex proteins in erythroid cells, with potential clinical applications in transfusion medicine.

Generation of transgene-free hematopoietic stem cells from human induced pluripotent stem cells.

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.

Human induced pluripotent stem cells can reach complete terminal maturation: in vivo and in vitro evidence in the erythropoietic differentiation model.

BACKGROUND: Human induced pluripotent stem cells offer perspectives for cell therapy and research models for diseases. We applied this approach to the normal and pathological erythroid differentiation model by establishing induced pluripotent stem cells from normal and homozygous sickle cell disease donors. DESIGN AND METHODS: We addressed the question as to whether these cells can reach complete erythroid terminal maturation notably with a complete switch from fetal to adult hemoglobin. Sickle cell disease induced pluripotent stem cells were differentiated in vitro into red blood cells and characterized for their terminal maturation in terms of hemoglobin content, oxygen transport capacity, deformability, sickling and adherence. Nucleated erythroblast populations generated from normal and pathological induced pluripotent stem cells were then injected into non-obese diabetic severe combined immunodeficiency mice to follow the in vivo hemoglobin maturation. RESULTS: We observed that in vitro erythroid differentiation results in predominance of fetal hemoglobin which rescues the functionality of red blood cells in the pathological model of sickle cell disease. We observed, in vivo, the switch from fetal to adult hemoglobin after infusion of nucleated erythroid precursors derived from either normal or pathological induced pluripotent stem cells into mice. CONCLUSIONS: These results demonstrate that human induced pluripotent stem cells: i) can achieve complete terminal erythroid maturation, in vitro in terms of nucleus expulsion and in vivo in terms of hemoglobin maturation; and ii) open the way to generation of functionally corrected red blood cells from sickle cell disease induced pluripotent stem cells, without any genetic modification or drug treatment.

Red blood cells from induced pluripotent stem cells: hurdles and developments.

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.

Unimpaired terminal erythroid differentiation and preserved enucleation capacity in myelodysplastic 5q(del) clones: a single cell study.

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.

Red blood cell generation from human induced pluripotent stem cells: perspectives for transfusion medicine.

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.

Ex vivo generation of human red blood cells: a new advance in stem cell engineering.

We describe a technological approach permitting the massive expansion of CD34(+) stem cells and their 100% conversion ex vivo into mature red blood cells (RBC). The protocol comprises three steps: a first step consisting of cell proliferation and induction of erythroid differentiation in a liquid medium without serum in the presence of growth factors (GF), a second based on a model reconstitution of the medullar microenvironment (ME) (human MSC or murine stromal cells) in the presence of GF, and a third in the presence of the ME alone, without any GF. This work highlights the impact of the ex vivo microenvironment on the terminal maturation of erythroid cells. A critical point is that the RBC generated in vitro have all the characteristics of functional native adult RBC. Moreover, this new concept of 'cultured RBC' (cRBC) is important for basic research into terminal erythropoiesis and has major clinical implications, especially in transfusion medicine. The three-step protocol can be adapted to use hematopoietic stem cells (HSC) from diverse sources: peripheral blood, bone marrow or cord blood.

Mesenchymal Stem Cell Administration Attenuates Colon Cancer Progression by Modulating the Immune Component within the Colorectal Tumor Microenvironment.

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.

Design and Validation of an Automated Process for the Expansion of Peripheral Blood-Derived CD34+ Cells for Clinical Use After Myocardial Infarction.

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.

Impact of genotype on survival of children with T-cell acute lymphoblastic leukemia treated according to the French protocol FRALLE-93: the effect of TLX3/HOX11L2 gene expression on outcome.

BACKGROUND: The prognostic value of the ectopic activation of TLX3 gene expression, a major oncogenetic event associated with pediatric T-cell acute lymphoblastic leukemia, is controversial. Likewise, the frequency and the prognostic significance in pediatric T-cell acute lymphoblastic leukemia of the newly characterized NUP214-ABL1 fusion transcript is not yet clear. DESIGN AND METHODS: Two hundred children with T-cell acute lymphoblastic leukemia were treated in the French FRALLE-93 study from 1993 to 1999. The expression of TLX3, TLX1 and SILTAL1 genes was analyzed in samples from 92 patients by real-time quantitative reverse transcriptase polymerase chain reaction. Most of these samples were further studied for NUP214-ABL1 and CALM-AF10 fusion transcripts. RESULTS: The median follow-up was 7.9 years. At 5 years the overall survival (+/- standard deviation, %) was 62 (+/-3%) and leukemia-free survival was 58 (+/-3%). Patients with T-cell acute lymphoblastic leukemia positive for TLX3 had a poorer survival compared to those with T-ALL negative for TLX3 (overall survival: 45+/-11% vs. 57+/-5%, p=0.049). In multivariate analysis, TLX3 expression was an independent adverse risk factor predicting relapse with a hazard ratio of 2.44 (p=0.017) and an overall survival with a hazard ratio of 3.7 (p=0.001). NUP214-ABL1 was expressed in 16.6% of patients with TLX3-positive T-ALL (3 of 18); all of the patients with this association died before completion of the treatment. SILTAL expression did not significantly affect the prognosis of patients with T-cell acute lymphoblastic leukemia. Only three of 92 patients expressed the TLX1 gene and all three are alive. CONCLUSIONS: TLX3 gene expression is an independent risk factor predicting poor survival in childhood T-cell acute lymphoblastic leukemia. When co-expressed with TLX3, NUP214-ABL1 transcripts may increase the risk of poor survival.

Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells.

We describe here the large-scale ex vivo production of mature human red blood cells (RBCs) from hematopoietic stem cells of diverse origins. By mimicking the marrow microenvironment through the application of cytokines and coculture on stromal cells, we coupled substantial amplification of CD34(+) stem cells (up to 1.95 x 10(6)-fold) with 100% terminal differentiation into fully mature, functional RBCs. These cells survived in nonobese diabetic/severe combined immunodeficient mice, as do native RBCs. Our system for producing 'cultured RBCs' lends itself to a fundamental analysis of erythropoiesis and provides a simple in vitro model for studying important human viral or parasitic infections that target erythroid cells. Further development of large-scale production of cultured RBCs will have implications for gene therapy, blood transfusion and tropical medicine.

Author Correction: Synergistic effect of human Bone Morphogenic Protein-2 and Mesenchymal Stromal Cells on chronic wounds through hypoxia-inducible factor-1 α induction.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Ex vivo production of human red blood cells from hematopoietic stem cells: what is the future in transfusion?

There is difficulty in obtaining adequate supplies of blood components, as well as disappointing performance of stabilized or recombinant hemoglobins, limited indications of oxygen transporters (perfluorocarbons), and slow development of "universal" red blood cells (RBCs). There is, therefore, a need for complementary sources of RBCs for transfusion. Thus, an attempt to generate erythroid cells in vitro makes good sense. We describe in this article a methodology permitting the massive ex vivo production of mature human RBCs having all the characteristics of native adult RBCs from hematopoietic stem cells of diverse origins: blood, bone marrow, or cord blood. This protocol allows both the massive expansion of hematopoietic stem cells/progenitors and their complete differentiation to the stage of perfectly functional mature RBCs. The levels of amplification obtained (10(5) to 2 x 10(6)) are compatible with an eventual transfusion application. We discuss in this article the state of the art of this new concept and evoke possible obstacles that need to be overcome to pass from a laboratory model to clinical practice. We analyze its possible indications in the medium and long term, discuss the economic aspects, and raise the question: Can we afford the luxury of developing this approach, one that could represent a considerable advance in blood transfusion?

Human erythroid cells produced ex vivo at large scale differentiate into red blood cells in vivo.

New sources of red blood cells (RBCs) would improve the transfusion capacity of blood centers. Our objective was to generate cells for transfusion by inducing a massive proliferation of hematopoietic stem and progenitor cells, followed by terminal erythroid differentiation. We describe here a procedure for amplifying hematopoietic stem cells (HSCs) from human cord blood (CB) by the sequential application of specific combinations of growth factors in a serum-free culture medium. The procedure allowed the ex vivo expansion of CD34+ progenitor and stem cells into a pure erythroid precursor population. When injected into nonobese diabetic, severe combined immunodeficient (NOD/SCID) mice, the erythroid cells were capable of proliferation and terminal differentiation into mature enucleated RBCs. The approach may eventually be useful in clinical transfusion applications.

Synergistic effect of human Bone Morphogenic Protein-2 and Mesenchymal Stromal Cells on chronic wounds through hypoxia-inducible factor-1 α induction.

Chronic skin ulcers and burns require advanced treatments. Mesenchymal Stromal Cells (MSCs) are effective in treating these pathologies. Bone Morphogenic Protein-2 (BMP-2) is known to enhance angiogenesis. We investigated whether recombinant human hBMP-2 potentiates the effect of MSCs on wound healing. Severe ulceration was induced in rats by irradiation and treated by co-infusion of MSCs with hBMP-2 into the ulcerated area which accelerated wound healing. Potentiation of the effect of MSCs by hBMP-2 on endothelial repair improved skin healing. HBMP-2 and MSCs synergistically, in a supra additive or enhanced manner, renewed tissue structures, resulting in normalization of the epidermis, hair follicles, sebaceous glands, collagen fibre density, and blood vessels. Co-localization of MSCs with CD31 + cells suggests recruitment of endothelial cells at the site of injection. HBMP-2 and MSCs enhanced angiogenesis and induced micro-vessel formation in the dermis where hair follicles were regenerated. HBMP-2 acts by causing hypoxia-inducible factor-1 α (HIF-1α) expression which impacts endothelial tube formation and skin repair. This effect is abolished by siRNA. These results propose that new strategies adding cytokines to MSCs should be evaluated for treating radiation-induced dermatitis, burns, and chronic ulcers in humans.