Myeloma is characterized by bone lesions, which are related to both an increased osteoclast activity and a defect in the differentiation of medullary mesenchymal stem cells (MSCs) into osteoblasts. ...Outside the medullary environment, adipocyte-derived MSCs (ASCs) could represent a source of functional osteoblasts. However, we recently found a defect in the osteoblastic differentiation of ASCs from myeloma patients (MM-ASCs). We examined the effects of plasma from myeloma patients at diagnosis (MM-plasmas) and in complete remission (CR-plasmas) and from healthy donors on the osteoblastic differentiation of healthy donor-derived ASCs (HD-ASCs). Osteoblastogenesis in HD-ASCs was suppressed by MM-plasmas. Seven cytokines (ANG1, ENA-78, EGF, PDGF-AA/AB/BB, and TARC) were increased in MM-plasmas and separately inhibited the osteoblastic differentiation of HD-ASCs. Comparison of MM-ASCs and HD-ASCs by RNA sequencing showed that two master genes characterizing adipocyte differentiation, CD36 and PPARγ, were upregulated in MM-ASCs as compared to HD-ASCs. Finally, we demonstrated a significant increase in CD36 and PPARγ expression in HD-ASCs in the presence of MM-plasmas or the seven cytokines individually, similarly as in MM-ASCs. We conclude that specific cytokines in MM-plasmas, besides the well-known DKK1, inhibit the osteoblastic differentiation of MM- and HD-ASCs with a skewing towards adipocyte differentiation.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
The 5q- syndrome should be considered a quantitative rather than qualitative bone marrow defect. This observation might open the way to new therapeutic concepts.
Introduction: Myeloma is characterized by bone lesions, which are related to both an increased osteoclast activity and a defect in the differentiation of medullary mesenchymal stem cells (MSCs) into ...osteoblasts. Outside the medullary environment, adipocyte-derived MSCs (ASCs) could represent a source of functional osteoblasts. However, we recently found a defect in the osteoblastic differentiation of ASCs from myeloma patients (MM-ASCs). We therefore examined the effects of plasma from myeloma patients at diagnosis (MM-plasmas) and in complete remission (CR-plasmas) and from healthy donors on the osteoblastic differentiation of healthy donor-derived ASCs (HD-ASCs) and healthy donor-bone marrow derived MSCs (HD-BM-MSCs). Materials and Methods: We studied 11 MM-ASCs, 5 HD-ASCs and 3 HD-BM-MSCs. The plasmas were from myeloma patients with bone lesions at diagnosis (n=12), in complete remission (n=8) and from 5 pools of 100 healthy donors (HD-plasmas). HD-ASCs were differentiated into osteoblasts and adipocytes and HD-BM-MSCs into osteoblasts with the three types of plasmas as well as newly discovered cytokines. Results: Osteoblastogenesis in HD-ASCs was suppressed by MM-plasmas. Alizarin red coloration and alkaline phosphatase activity were strongly decreased along with a decreased RUNX2 and osteocalcin expression. However, adipocyte differentiation was unaltered. The osteoblastic differentiation deficiency was reversible once the plasma-derived factors were removed. Using cytokine array and comparing MM-plasmas with HD-plasmas, we identified seven cytokines (ANG1, ENA-78, EGF, PDGF-AA/AB/BB and TARC), besides DKK1, highly increased in MM-plasmas which was confirmed by ELISA (Figure). They separately inhibited the osteoblastic differentiation of HD-ASCs. In contrast, myeloma patients in remission had a cytokine plasma level almost normal with barely no osteoblastic differentiation inhibition. In addition, the mixture of the 7 cytokines with and without DKK1 inhibited not only the HD-ASCs but also the HD-BM-MSCs. Concomittantly, we observed that MM-plasmas enhanced adipogenesis-related gene expression. Comparison of MM-ASCs and HD-ASCs by RNA sequencing showed that two master genes characterizing adipocyte differentiation, CD36 and PPARγ, were upregulated in MM-ASCs as compared to HD-ASCs. Moreover, we demonstrated a significant increase in CD36 and PPARγ expression in HD-ASCs in the presence of MM-plasmas or the seven cytokines individually, similarly as in MM-ASCs. Finally, we tried to identify the origin of these cytokines. When myeloma patients were in remission, the cytokines levels were strongly decreased suggesting a malignant plasmocyte secretion. This was reinforced by the detection of the 7 cytokines in three different myeloma cell lines with an especially high secretion of PDGF-AA. We conclude that specific cytokines in MM-plasmas, besides the well-known DKK1, inhibit the osteoblastic differentiation of MM- and HD-ASCs with a skewing towards adipocyte differentiation. Of note, this inhibition by the cytokines were also observed on HD-BM-MSCs suggesting that this could also be the case on myeloma-BM-MSCs.
Legend to figure: Cytokine expression in MM, CR and HD-plasmas
(A) Representative images of cytokine array blots probed with the plasma samples. The red boxes identify the cytokines significantly dysregulated in MM- as compared to HD- or CR-plasmas and further analyzed by ELISA. The blue boxes identified the cytokines similarly expressed in MM-/CR-/HD-plasmas. (B) Cytokine concentrations in the HD-plasmas (n=5), MM-plasmas (n=11) and CR-plasmas (n=8) were measured by ELISA. * p < 0.05, ** p < 0.01, *** p < 0.001, ns (not significant).
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Delhommeau: Novartis: Consultancy; BMS: Consultancy; Celgene: Consultancy. Garderet: Celgene: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Sanofi: Consultancy; Takeda: Consultancy.
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.
Myeloma is characterized by bone lesions, which are related to both an increased osteoclast activity and a defect in the differentiation of medullary mesenchymal stem cells (MSCs) into osteoblasts. ...Outside the medullary environment, adipocyte-derived MSCs (ASCs) could represent a source of functional osteoblasts. However, we recently found a defect in the osteoblastic differentiation of ASCs from myeloma patients (MM-ASCs). We aimed to identify the cause of this defect.
We examined the effects of plasma from myeloma patients at diagnosis (MM-plasmas), in complete remission (CR-plasmas) and from healthy donors on the osteoblastic differentiation of MM-ASCs, healthy donor-derived ASCs (HD-ASCs) and healthy donor-bone marrow derived MSCs (HD-BM-MSCs). Alizarin red coloration, alkaline phosphatase activity, RUNX2 and osteocalcin expression determined osteoblastic differentiation while oil-red O staining, PPARγ, C/EBPα and CD36 expression reflected the adipogenic capacity. Cytokine arrays were used along with Elisa assays for identification and quantification of relevant cytokines. RNA sequencing was performed on MM and HD-ASCs.
Osteoblastogenesis in HD-ASCs was suppressed by MM-plasmas but adipocyte differentiation was unaltered. This defect was reversible once the plasma-derived factors were removed. Using cytokine array and comparing MM-plasmas with HD-plasmas, we identified seven cytokines (ANG1, ENA-78, EGF, PDGF-AA/AB/BB and TARC), besides DKK1, highly increased in MM-plasmas. They separately inhibited the osteoblastic differentiation of HD-ASCs. In contrast, in CR-plasmas, the cytokine plasma levels were almost normal with barely no osteoblastic differentiation inhibition. In addition, the mixture of the 7 cytokines with and without DKK1 inhibited not only the HD-ASCs but also the HD-BM-MSCs. Concomitantly, MM-plasmas enhanced adipogenesis-related gene expression. Comparison of MM-ASCs and HD-ASCs by RNA sequencing showed that two master genes characterizing adipocyte differentiation, CD36 and PPARγ, were upregulated in MM-ASCs as compared to HD-ASCs. Moreover, we demonstrated a significant increase in CD36 and PPARγ expression in HD-ASCs in the presence of MM-plasmas or the seven cytokines individually, similarly as in MM-ASCs. Finally, we tried to identify the origin of these cytokines. In CR-plasmas, the cytokine levels were strongly decreased suggesting a malignant plasmocyte secretion. This was reinforced by the detection of the 7 cytokines in three different myeloma cell lines with an especially high secretion of PDGF-AA.
We conclude that specific cytokines in MM-plasmas, besides the well-known DKK1, inhibit the osteoblastic differentiation of MM- and HD-ASCs with a skewing towards adipocyte differentiation. Of note, this inhibition, by these new circulating cytokines, was also observed on HD-BM-MSCs, suggesting that this could also be the case on myeloma-BM-MSCs.
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