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.

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.

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.

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.

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.

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.

Genetic hierarchy and temporal variegation in the clonal history of acute myeloid leukaemia.

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.

Comprehensive Proteomic Analysis of Human Erythropoiesis.

Mass spectrometry-based proteomics now enables the absolute quantification of thousands of proteins in individual cell types. We used this technology to analyze the dynamic proteome changes occurring during human erythropoiesis. We quantified the absolute expression of 6,130 proteins during erythroid differentiation from late burst-forming units-erythroid (BFU-Es) to orthochromatic erythroblasts. A modest correlation between mRNA and protein expression was observed. We identified several proteins with unexpected expression patterns in erythroid cells, highlighting a breakpoint in the erythroid differentiation process at the basophilic stage. We also quantified the distribution of proteins between reticulocytes and pyrenocytes after enucleation. These analyses identified proteins that are actively sorted either with the reticulocyte or the pyrenocyte. Our study provides the absolute quantification of protein expression during a complex cellular differentiation process in humans, and it establishes a framework for future studies of disordered erythropoiesis.

[In vitro generation of blood red cells from stem cells: a sketch of the future]. / Génération de globules rouges de culture à partir de cellules souches : bref récit du futur.

Human adult pluripotent stem cells, stem cells of embryonic origin and induced pluripotent stem cells (iPS) provide cellular sources for new promising regenerative medicine approaches. Because these cells can be patient-specific, they allow considering a personalized medicine appropriate to the diagnosis of each. The generation of cultured red blood cells (cRBC) derived from stem cells is emblematic of personalized medicine. Indeed, these cells have the advantage of being selected according to a blood phenotype of interest and they may provide treatments to patients in situation of impossible transfusion (alloimmunized patients, rare phenotypes). Essential progresses have established proof of concept for this approach, still a concept some years ago. From adult stem cells, all steps of upstream research were successfully achieved, including the demonstration of the feasibility of injection into human. This leads us to believe that Red Blood Cells generated in vitro from stem cells will be the future players of blood transfusion. However, although theoretically ideal, these stem cells raise many biological challenges to overcome, although some tracks are identified.

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.

Long-Term Quantitative Biodistribution and Side Effects of Human Mesenchymal Stem Cells (hMSCs) Engraftment in NOD/SCID Mice following Irradiation.

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.

Large-scale production of red blood cells from stem cells: what are the technical challenges ahead?

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.

Human mesenchymal stem cells provide protection against radiation-induced liver injury by antioxidative process, vasculature protection, hepatocyte differentiation, and trophic effects.

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.

New insights for pelvic radiation disease treatment: Multipotent stromal cell is a promise mainstay treatment for the restoration of abdominopelvic severe chronic damages induced by radiotherapy.

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.

Fifteen years of preclinical and clinical experiences about biotherapy treatment of lesions induced by accidental irradiation and radiotherapy.

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.

Caspase-3 is involved in the signalling in erythroid differentiation by targeting late progenitors.

A role for caspase activation in erythroid differentiation has been established, yet its precise mode of action remains elusive. A drawback of all previous investigations on caspase activation in ex vivo erythroid differentiation is the lack of an in vitro model producing full enucleation of erythroid cells. Using a culture system which renders nearly 100% enucleated red cells from human CD34(+) cells, we investigated the role of active caspase-3 in erythropoiesis. Profound effects of caspase-3 inhibition were found on erythroid cell growth and differentiation when inhibitors were added to CD34(+) cells at the start of the culture and showed dose-response to the concentration of inhibitor employed. Enucleation was only reduced as a function of the reduced maturity of the culture and the increased cell death of mature cells while the majority of cells retained their ability to extrude their nuclei. Cell cycle analysis after caspase-3 inhibition showed caspase-3 to play a critical role in cell proliferation and highlighted a novel function of this protease in erythroid differentiation, i.e. its contribution to cell cycle regulation at the mitotic phase. While the effect of caspase-3 inhibitor treatment on CD34(+) derived cells was not specific to the erythroid lineage, showing a similar reduction of cell expansion in myeloid cultures, the mechanism of action in both lineages appeared to be distinct with a strong induction of apoptosis causing the decreased yield of myeloid cells. Using a series of colony-forming assays we were able to pinpoint the stage at which cells were most sensitive to caspase-3 inhibition and found activated caspase-3 to play a signalling role in erythroid differentiation by targeting mature BFU-E and CFU-E but not early BFU-E.

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.

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.