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Clonal history of a cord blood donor cell leukemia with prenatal somatic JAK2 V617F mutation

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.

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.

The induction of micronucleated reticulocytes in the bone marrow is a sensitive indicator of chromosomal damage. Therefore, the micronucleus assay in rodents is widely used in genotoxicity and carcinogenicity testing. A test system based on cultured human primary cells could potentially provide better prediction compared to animal tests, increasing patient safety while also implementing the 3Rs principle, i.e. replace, reduce and refine. Hereby, we describe the development of an in vitro micronucleus assay based on animal-free ex vivo culture of human red blood cells from hematopoietic stem cells. To validate the method, five clastogens with direct action, three clastogens requiring metabolic activation, four aneugenic and three non-genotoxic compounds have been tested. Also, different metabolic systems have been applied. Flow cytometry was used for detection and enumeration of micronuclei. Altogether, the results were in agreement with the published data and indicated that a sensitive and cost effective in vitro assay to assess genotoxicity with a potential to high-throughput screening has been developed. (C) 2015 The Authors. Published by Elsevier Ltd.

We examined whether ex vivo expansion of umbilical cord blood progenitor cells affected their capacity to generate immune cells such as T lymphocytes (TLs) and dendritic cells (DCs). The capacity to generate TLs from cord blood CD34(+) cells expanded for 14 days (d14) was compared with that of nonexpanded CD34(+) cells (d0) using fetal thymus organ cultures or transfer into nonobese diabetic/severe combined immunodeficient mice. The cell preparations yielded comparable percentages of immature (CD4(+)CD8(-), CD4(+)CD8(+)) TLs and functional mature (CD3(+)CD4(+), CD3(+)CD8(+)) TLs with an analogous TCR (T-cell receptor)-Vbeta repertoire pattern. As regards DCs, d0 and d14 CD34(+) cells also yielded similar percentages of CD1a(+) DCs with the same expression levels of HLA-DR, costimulatory and adhesion molecules, and chemokine receptors. DCs derived from either d14 or d0 CD34(+) stimulated allogeneic TLs to the same extent, and the cytokine pattern production of these allogeneic TLs was similar with no shift toward a predominant Th1 or Th2 response. Even though the intrinsic capacity of d14 CD34(+) cells to generate DCs was 13-fold lower than that of d0 CD34(+) cells, this reduction was offset by the prior amplification of the CD34(+) cells, resulting in the overall production of 15-fold more DCs. These data indicate that ex vivo expansion of CD34(+) cells does not impair T lymphopoiesis nor DC differentiation capacity.

We describe a technical approach permitting massive expansion of CD34+ stem cells (up to 1.95 x 10(6)-fold) and their full ex vivo conversion into mature red blood cells (RBCs). This three-step protocol can be adapted to hematopoietic stem cells (HSC) of various origins. First, cell proliferation and erythroid differentiation are induced in serum-free media supplemented with stem cell factor, interleukin-3 and erythropoietin (Epo) for 8 days. The cells are then co-cultured with either the murine stromal cell line MS-5 or human mesenchymal cells for 3 days in the presence of Epo alone. Finally, all exogenous factors are withdrawn and the cells are incubated on a simple stroma for up to 10 days. The ex vivo microenvironment strongly influences both the terminal maturation of erythroid cells and hemoglobin (Hb) synthesis. Critically, in vitro-generated RBCs have all the characteristics of functional native adult RBCs in terms of their enzyme content, membrane deformability, and capacity to fix and release oxygen. In addition, their behavior in the murine NOD/SCID model mirrors that of native RBCs. This new concept of "cultured RBCs" (cRBC) has major implications for basic research on terminal erythropoiesis and for patient management. Currently, the potential yield of functional red cells is compatible with clinical requirements, as several units of packed RBCs can be produced from a single donation. Importantly, infused cRBC would all have a life-span of about 120 days, whereas the mean half-life of normal donor RBCs is only 28 days. This would help to minimize the transfusion exposure of patients requiring regular treatment, thereby reducing the risk of iron overload and allo-immunization. The use of autologous CD34+ cells isolated from leukapheresis samples could be beneficial for patients who no longer tolerate allogeneic RBCs. This new method should also prove useful for analyzing the mechanisms of terminal erythropoiesis, including hemoglobin synthesis. Finally, it could provide a tool for investigating the lifecycle of blood parasites such as Plasmodium, the agent of malaria.

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.

The aim of the present report is to describe clinically relevant culture conditions that support the expansion of primitive hematopoietic progenitors/stem cells, with maintenance of their hematopoietic potential as assessed by in vitro assays and the NOD-SCID in vivo repopulating capacity.CD34(+) cord blood (CB) cells were cultured in serum-free medium containing stem cell factor, Flt3 ligand, megakaryocyte growth and development factor, and granulocyte colony-stimulating factor. After 14 days, the primitive functions of expanded and nonexpanded cells were determined in vitro using clonogenic cell (colony-forming cells, long-term culture initiating cell [LTC-IC], and extended [E]-LTC-IC) and lymphopoiesis assays (NK, B, and T) and in vivo by evaluating long-term engraftment of the bone marrow of NOD-SCID mice. The proliferative potential of these cells also was assessed by determining their telomere length and telomerase activity. Levels of expansion were up to 1,613-fold for total cells, 278-fold for colony-forming unit granulocyte-macrophage, 47-fold for LTC-IC, and 21-fold for E-LTC-IC. Lymphoid B-, NK, and T-progenitors could be detected. When the expanded populations were transplanted into NOD-SCID mice, they were able to generate myeloid progenitors and lymphoid cells for 5 months. These primitive progenitors engrafted the NOD-SCID bone marrow, which contained LTC-IC at the same frequency as that of control transplanted mice, with conservation of their clonogenic capacity. Moreover, human CD34(+)CDl9(-) cells sorted from the engrafted marrow were able to generate CD19(+) B-cells, CD56(+)CD3(-) NK cells, and CD4(+)CD8(+)alphabetaTCR(+) T-cells in specific cultures. Our expansion protocol also maintained the telomere length in CD34(+) cells, due to an 8.8-fold increase in telomerase activity over 2 weeks of culture. These experiments provide strong evidence that expanded CD34(+) CB cells retain their ability to support long-term hematopoiesis, as shown by their engraftment in the NOD-SCID model, and to undergo multilineage differentiation along all myeloid and the B-, NK, and T-lymphoid pathways. The expansion protocol described here appears to maintain the hematopoietic potential of CD34(+) CB cells, which suggests its relevance for clinical applications.

The aim of the present study was to evaluate the capacity to expand of hematopoietic stem cell (HSC) samples from eight patients with NHL, and to follow in parallel the fate of tumor cells in four of eight samples still containing bcl2/JH+ tumor cells after CD34+ or CD19-/20-/34+ cell selection. The presence of bcl2/JH+ cells was also investigated after expansion in four of eight samples, two of which were bcl2/JH at harvesting and two which were initially bcl2/JH+ but became bcl2/JH (below the level of PCR detection) after cell selection, to assess a possible reappearance of occult tumor cells after expansion culture. We used culture conditions that we previously had established to allow high level expansion of normal precursors, progenitors and LTC-ICs. In this study, particular attention was given to the role of Flt3-ligand, known to favor the growth of B cells. The expansion conditions were: 1.5 x 10(3) cells/ml in serum-free medium containing stem cell factor (SCF), interleukin-3 (IL-3), IL-6, granulocyte-stimulating factor (G-CSF), erythropoietin (Epo) +/- Flt3-ligand (Flt3-L) for 10 days. After culture, total cells, CFU-GMs, BFU-Es and LTC-ICs were expanded to a mean of 833-, 6.6-, 4.6-, and 1.8-fold, respectively with the cocktail of cytokines not including Flt3-L. When Flt3-L was added, the mean expansion values were 1095-, 31-, 15- and three-fold, respectively. Residual bcl2/JH+ cells present in four of eight samples before expansion were not detected after expansion. Similarly, no tumor cells reappeared after expansion of the two samples which had become negative after selection, as well as in the two samples which were bcl2/JH- at harvesting. These results suggest first that ex vivo expansion of hematopoietic stem cells in patients with non-Hodgkin's lymphoma is feasible without incurring the parallel risk of amplifying tumor cells; second, that Flt3-L did not stimulate the growth of tumor cells while it clearly favored the growth of normal progenitors.

Several prospective randomized trials in acute myelocytic leukemia (AML) documented a lower relapse rate with autologous bone marrow transplantation (ABMT) than with conventional chemotherapy. However, they also identified some transplant difficulties, such as failure to collect sufficient numbers of stem cells, slow kinetics of engraftment, and a high transplant-related mortality that diminished or negated positive impact on overall survival. Data for ABMT are inconclusive in acute lymphocytic leukemia (ALL) in adults. We retrospectively analyzed patients with acute leukemia autografted with marrow purged with mafosfamide after January 1983 in our institution. The population comprised 229 consecutive patients; 165 with AML [123 in first remission (CR1), 32 in second remission (CR2)]; 61 with ALL (46 in CR1, 4 in CR2); and 3 with undifferentiated acute leukemia. All patients were autografted with marrow purged with mafosfamide. Mafosfamide was given at a constant dose of 50 μg/mL in 103 and adjusted individually to produce a CFU-GM LD 95 (5% residual CFU-GM post purging) in 126. The outcome was analyzed for correlation with patient characteristics, the disease including cytogenetics, and the graft itself. Prognostic factors identified by multivariate analysis were used to derive a prognostic classification. Patients receiving higher doses of marrow submitted to purging (>5.46 × 10 4 CFU-GM/kg) experienced a lower treatment-related mortality (RR = 0.11, p=0.005) and a higher leukemia-free (RR = 0.5, p=0.005) and overall survival (RR = 0.4, p=0.001). Patients receiving 5.46 × 10 4 CFU-GM/kg and doses actually infused post purging of ≤0.02 × 10 4 /kg had a treatment-related mortality of only 2 ± 2%, a leukemia-free survival of 70%, and an overall survival of 77 ± 7% at 10 years. In this study of autotransplantation for acute leukemia using mafosfamide-purged marrow, the stem cell dose used for purging and the intensity of purging were the most important factors predicting outcome.

The aim of this study was to evaluate the ex vivo expansion of normal CD34 + cells in gas-permeable polypropylene bags suitable for clinical use. Cells were cultured for 14 days in serum-free medium supplemented with SCF, IL3, IL6, FLT3-1, G-CSF ± MGDF or Epo. The bags supported the expansion of hematopoietic cells in a similar manner to small scale well or flask systems, allowing mean expansions of up to 2193-fold for total nucleated cells, 140-fold for CFU-GM and 66-fold for LTC-IC. Increasing the initial cell concentration from 5 x 10 3 to 1 x 10 5 CD34 + cells/ml induced the production of granulocytic cells with terminal differentiation while simultaneously decreasing the overall extent of expansion of the white blood cells produced. We tested the phagocytic activity and oxidative metabolism of the white blood cells produced. The percentage of phagocytic cells was 39 ± 0.5% in expanded cultures derived from fractions initiated at 5 x 10 3 , 104 or 10 5 cells/ml and 45 ± 6% in cultured cells obtained from starting fractions containing 5 x 10 4 cells/ml, as compared to 58 ± 4% in normal controls. A study of the potential for oxygen-dependent microbe killing showed that the expanded cells produced H 2 O 2 , although in lesser quantities than control cells. We subsequently investigated the possibility of freezing expanded cells. Total cell recovery after thawing was 45 ± 4%, while recoveries of progenitors and stem cells ranged from 65 to 90%, without any influence of the initial cell concentration. This new approach could be of major interest for clinical practice, as it would allow evaluation of the quality of a graft prior to its infusion and employs experimental conditions which meet the criteria for potential clinical use.

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.

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.

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.

To circumvent the problems inherent in allogeneic bone marrow transplantation, allogeneic cord blood has been studied as an alternative source of hematopoietic stem cells. Preliminary results with cord blood from an HLA-matched sibling are encouraging, but the experience with this procedure is limited.1–5 Although some 200 transplantations of cord blood have been performed in children,6,7 few have been performed in adults. We describe an adult with chronic myelogenous leukemia who underwent the transplantation of cord-blood stem cells successfully. Case Report A 26-year-old woman was found to have chronic myelogenous leukemia in July 1990. All the leukemic cells had . . .

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.

International audience; 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. © 2012 Ferrata Storti Foundation.

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 1010 cRBCs generated under good manufacturing practice conditions and labeled with 51Cr. 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 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.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.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.

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

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.

The work on human embryonic stem cells is being performed with a variety of cell lines using a variety of culture conditions; a situation that makes standardisation between projects and publications very difficult. Clearly the consequence of using such cells would be wasted time and resources but, more seriously, the generation of erroneous data in the literature which could both confuse and delay scientific progress in this area. This guidance document represents the outcome of the first meeting of the group named The International Stem Cell Banking Initiative held in October 2007. The document has been prepared from the perspective of hESC culture but, in many respects, is broadly applicable to all human stem cell lines including induced pluripotent stem cell lines.

We have sufficient knowledge of the biology of hematopoietic stem cells to hope that we might generate human red blood cells in the laboratory. It may soon be possible to produce enough to transfuse "cultured" red blood cells to manufacture human red blood cells from hematopoietic stem cells for transfusion purposes. This article describes progress and the challenges that remain in the search for in vitro generated red blood cells that can be efficiently manufactured in high volumes and given to any recipient.

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.

For several years, researchers have been trying to find a substitute for red blood cells (RBC). The development of chemical or natural molecules to replace hemoglobin has nevertheless proved difficul

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?

Peripheral blood CD34+ cell samples from eight children with advanced neuroblastoma and from 10 healthy adult donors were seeded at 5 x 10(4) cells/ml in stroma-free, serum-free medium with FL, SCF, MGDF (100 ng/ml each), G-CSF, IL6 (10 ng/ml each) and IL3 (5 ng/ml), and incubated for 10 days. The levels of expansion of PBCD34+ cells observed in neuroblastoma patients, with up to 214-fold expansion for total nucleated cells, 39-fold for CD34+ cells, 79-fold for CFU-GM and nine-fold for LTC-IC were identical to those obtained with PBCD34+ cells of healthy donors (P/=0.5). All samples from patients with neuroblastoma and five donor's PBCD34+ cell samples contaminated with IMR-32 neuroblasts, were screened for the number of tyrosine hydroxylase (TH) mRNA transcript using LightCycler software. In all samples, progressive 1.9-4.4 log decreases in the number of TH transcripts were observed between days 0 and 10 of expansion. Our results show that in extensively pretreated children with neuroblastoma, the culture conditions that were effective for BM and CB cell expansion can generate an expansion of PBCD34+ cells and provide a purge of tumour cells.

In vitro production of red blood cells (RBCs) from stem cells (SCs) for transfusion is emblematic of what regenerative medicine could be in the near future [1]. This project of proposing an alternative to conventional transfusion, includes four challenges – scientific, technological, public health and social – that must be overcome to attain large-scale clinical development. First, a scientific challenge: recreate in vitro a fundamental physiological mechanism – the complete and terminal maturation of RBCs from a SC population. The goal is to reach the ultimate stage of the erythroid lineage, cells that specialize in oxygen transport to the point of losing all other functions, even eliminating their nucleus. The complexity of this concept resides in that we need to identify the best source of SCs to attain a double goal: qualitative (mature and functional cells) and quantitative (massive amplification allowing transfusion applications). When this project was initiated in early 2000, the target population was limited to hematopoietic SCs from bone marrow, blood or umbilical cord. Today, other major SCs are putative candidates, the pluripotent, embryonic SCs or adults SCs (induced pluripotent SCs; iPSCs) [2]. Immortalized lines of CD34 cells will probably soon be available also. Indeed, while hematopoietic SCs theoretically allow our quantitative goal of RBC production for transfusion to be reached, their availability is dependent on donors and is thus limited. These are the limitations. Because iPSCs can proliferate indefinitely, they are potential candidates to provide complementary sources of RBCs for transfusion. Proof of concept for generating human RBCs from SCs has been performed, but the procedures need to be optimized to lead to clinical application in blood transfusion [3]. Several crucial points remain to be resolved. Notably, these include: the choice of the initial cell type to generate iPSCs; the method of reprogramming to ensure their safety as clinical-grade cells; the optimization of erythrocyte differentiation; and the definition of GMP conditions for industrial production. Thus, while we know the goal to attain, we do not know yet from where to start. It is a paradox! Fundamental science moves faster than industrial development. This problem is common to all fields of regenerative medicine. Let us take the example of cardiac regeneration. Who can say today which is the appropriate target: total bone marrow cells, hematopoietic CD34 cells, CD133 cells, mesenchymal SCs, endothelial cells, embryonic SCs or iPSCs? All leads must be investigated without exclusion or bias.

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.

The ex vivo expansion of hematopoietic stem cells (HSC) for clinical use is now recognized to be a feasible and very promising approach for hematotherapy. Expansion of specific HSC subsets is required for different clinical applications, for example, to increase the number of mature cells, to produce specific cells for adoptive therapy, or to increase the number of primitive stem cells available for engraftment. Although hematopoietic growth factors can play an important role in this setting, in this review we emphasize that other variables affect the outcome of stem and progenitor cell expansion. These variables include the serum supplement, the purity of CD34(+) cells, the initial cell concentration, and the duration of culture. It is also essential to define standard culture conditions for normal stem cells and to limit or prevent expansion of residual tumor cells. In clinical applications, determination of the hematopoietic value of the expanded population is mandatory. Thus, we have to demonstrate the expansion of primitive hematopoietic progenitor and stem cells, with maintenance of their hematopoietic potential as assessed by in vitro or in vivo assays. We draw attention to the challenges in the clinical application of ex vivo expansion. These include the establishment of well-defined experimental conditions and the determination of the hematopoietic value of the expanded grafts, whatever the graft source: bone marrow, mobilized peripheral blood, or cord blood. Future studies hopefully will optimize these procedures and allow not only expansion but engineering of defined cellular functions as HSCs grow under defined conditions.

We tested two positive selection techniques for separation of CD34+ cells from bone marrow and analyzed the yields of CD34+ cells, BFU-E, CFU-GM, CFU-MK and LTC-IC after selection and expansion. An immunoadsorption procedure (CellPro) and an immunomagnetic (Baxter) CD34+ cell separation method were employed to purify the same bone marrow samples from seven normal subjects. Mean yields of CFU-GM and CFU-MK and absolute numbers of LTC-ICs were not different in the two purified cell populations. In contrast, the mean recovery of BFU-E was significantly lower for the immunoadsorption (21 +/- 14%) than for the immunomagnetic technique (44 +/- 27%). After separation, CD34+ cells were evaluated in 10-day liquid cultures for their expansion capacity in terms of total cells and progenitors. The expansion capacity of progenitors such as CFU-GM, CFU-MK and especially BFU-E selected by immunoadsorption was higher than the capacity of progenitors obtained by immunomagnetism, although final total and progenitor cell numbers are similar. Our results suggest that the populations separated by the two techniques differ mainly in the expansion capacity of progenitors and in the recovery of BFU-E after the selection procedure. These differences between two methods, which already are widely employed in research and in clinical transplantation, should be taken into account when considering the aims of the experiments.

The aim of the study is to define the ability of Flt3 ligand, MGDF, Epo and G-CSF to modulate the expansion of different hematopoietic compartments in association with a basic cocktail of SCF + IL-3 + IL-6 (S36). CD34+ cells from normal bone marrow were cultured in stroma-free, serum-free medium for 10 days. Using various concentrations of cytokines, total cells could be expanded up to 5200-fold, CD34+ cells up to 78-fold, CFU-GM up to 143-fold, BFU-E up to 46-fold, CFU-MK up to six-fold and LTC-IC up to four-fold. The results were assessed by multiparametric analysis of variance. Three factors had a significant stimulatory effect on the late precursor compartment: Epo (P < 10(-5)), G-CSF (P=5 x 10(-3)) and FL (P=10(-5)). Two were critical for CD34+ cell expansion: FL (P=4 x 10(-5)) and Epo (P=6 x 10(-5)), while two were critical for BFU-E expansion: MGDF (P=8 x 10(-4)) and FL (P=0.017). FL strongly stimulated CFU-GM expansion (P < 10(-5)), whereas none of the growth factors studied had any effect on CFU-MK. FL (P=10(-4)) and MGDF (P=0.002) were essential to obtain high levels of expansion of LTC-IC as determined in limiting dilution assays. In the light of the above results showing a preferential effect on the expansion of precursor cells (3080-fold), CD34+ cells (53-fold), CFU-GM (134-fold), BFU-E (46-fold) and LTC-IC (five-fold), the combination SCF, IL-3, IL-6, FL, MGDF, Epo and G-CSF was chosen as a putative cytokine cocktail for further studies on long-term culture. Sustained production of precursor cells, progenitor cells, LTC-IC and E-LTC-IC for up to 100 days reflects the persistence of very primitive stem cells. This suggests that these populations are probably able to undergo self-renewal divisions. The above combination of cytokines meets the required criterion for potential clinical application, which may be defined as an effective capacity to expand all cell compartments, using as the starting material high concentrations of low purity CD34+ cells.

We compared the effects of amifostine (WR-2721) on the cytotoxicity of mafosfamide or 4-hydroperoxycyclophosphamide (4-HC) in normal marrow progenitor cells (CFU-GM) and leukaemic progenitor cells (CFU-L) during ex vivo purging for autologous bone marrow transplantation (ABMT). Mononuclear cells (MNC) were incubated with amifostine 3 mg/ml for 15 min, washed, and subsequently tested for their sensitivity to mafosfamide or 4-HC (20-200 micrograms/ 10(7) MNC/ml). The LD95 was significantly higher among amifostine-treated cells for PCM-CFU-GM in 6 of 13 patients and for 5R-CFU-GM in 4 of 10 patients (P0.05). In contrast, amifostine exhibited no protective effects upon CFU-L. The results of this study will show that amifostine protects normal late and early progenitor cells for the toxic effects of cyclophosphamide derivatives while preserving their antileukaemic effects. These results suggest that amifostine has therapeutic value as a protective agent for normal marrow progenitor cells during ex-vivo purging of bone marrow for ABMT.

Abstract SCI-39 The generation of red blood cells (RBCs) in vitro using biotechnologies could represent an interesting alternative to classical transfusion products, in that it would combine adequate supplies with the specific production of blood products of a particular phenotype and the reduction of infection risks. This presentation will review how it is now possible to obtain in vitro complete maturation of the erythroid line to the stage of enucleation, starting from hematopoietic stem cells (HSCs) from peripheral blood, bone marrow or umbilical cord blood, or embryonic stem cells or adult pluripotent stem cells (induced pluripotent stem cells, iPSCs). This presentation will discuss how the functionality of cultured human RBCs (cRBCs) is settled in terms of deformability, hemoglobin maturation, oxygen carrying capacity, enzyme content, and terminal maturation from the reticulocyte stage to mature RBC after infusion into the NOD/SCID mouse model. The clinical feasibility of this concept has recently been demonstrated by reporting that cRBCs generated in vitro from peripheral HSCs under GMP conditions encounter in vivo the conditions required for their maturation and that they persist in the circulation for several weeks in humans. These data have established the proof of principle for transfusion of in vitro-generated RBCs and the pathway toward new developments in transfusion medicine. The most proliferative source of stem cells for generating cRBCs is cord blood, but it is limited in terms of HSCs and is dependent on donations. Pluripotent stem cell technology represents a potentially unlimited source of RBCs and opens the door to the development of a new generation of allogeneic transfusion products. Because iPSCs can be selected for a phenotype of interest, they are obviously the best candidate for organizing complementary sources of RBCs for transfusion. It is established that only three human iPSC clones would have been sufficient to match more than 99 percent of the patients in need of RBC transfusions. As a whole, a very limited number of RBC clones would provide for the needs of most alloimmunized patients and those with a rare blood group. Generating cRBCs from iPSCs has been done but needs to be optimized to lead to a clinical application in blood transfusion. Several crucial points remain to be resolved, notably, the choice of the initial cell type, the method of reprogramming (i.e., to ensure the safety of the iPSCs and to ensure their clinical grade), the optimization of the erythrocyte differentiation, and the definition of GMP conditions for industrial production. Assuming that in vitro large-scale cultured RBC production efficiently operates in the near future, this presentation will highlight the potential applications for alloimmunized patients and those with a rare blood group. Disclosures: No relevant conflicts of interest to declare.

We report a technological approach permitting, for the first time, the massive (up to 2x106-fold cell expansion) and selective (100%) ex vivo production of mature RBCs (cRBCs) starting from CD 34+ cells from peripheral blood (PB), bone marrow (BM) or cord blood (CB) into mature red cells in three steps: firstly, cell proliferation and erythroid differentiation were induced in serum free media supplemented with SCF, IL-3 and Epo for 8 days. Secondly, cells were co-cultured with additional Epo alone on either the murine MS-5 stromal cell line or human mesenchymal cells for 3 days. In the third step, all exogenous factors were withdrawn and cells were incubated on a simple stroma for 4 to 10 days. These cultured erythroid cells (reticulocytes and mature RBCs) displayed characteristics identical to those of native cells, in terms of MCV, MCH, MCHC, enzyme content (G6PD and PK) and deformability. The nature of the Hb produced depended on both the origin of the CD34+ cells and the culture conditions. cRBCs derived from PB or adult BM contained adult Hb (95±1%) whereas cRBCs derived from CB contained essentially HbF (64±13%). As for native RBCs, Hb was able to fix and release oxygen. CFSE-labelled-reticulocytes ex vivo generated from leukapheresis were injected into NOD-SCID mice. The transfused reticulocytes were found in the circulation to the same extent as native RBCs and fully matured into RBCs. This methodology is applicable for fundamental analysis of the mechanisms of terminal erythropoiesis and hemoglobin synthesis. Moreover, large scale cRBCs production could be possible with such a protocol. It can therefore be extrapolated to a wide range of clinical applications in the field of gene therapy, infectious diseases and particularly transfusion medicine with a pointed interest for the generation of a cell population homogeneous in age, thus achieving the new concept of cultured RBCs transfusion.

Hematopoietic Stem Cell Protocols (Methods in Molecular Medicine)edited by Christopher A. Klug and Craig T. Jordan. Humana Press, 2001.The past decade has produced remarkable advances in our ability to characterize the various cell lineages of human hematopoietic stem cells (HSCs). HSCs are self-renewing progenitors that give rise to all lineages of blood cells. They are found in all hematopoietic organs, from para-aortic mesoderm and yolk sac in fetuses to the bone marrow, blood and spleens of adults. Our knowledge of HSCs can be applied successfully in clinical practice. The most obvious immediate benefit is the possibility of increasing the chemotherapeutic dose in cancer and hematological malignancies by way of hematopoietic stem cell transplantation. This technique has potential in many clinical settings, including the treatment of other non-malignant diseases.Medical knowledge is constantly renewed. This is particularly true for hematopoietic stem-cell transplantation, hematopoietic gene therapy, solid organ transplantation and somatic tissue regeneration. The technology involved is developing rapidly and in this context the selection of stem cells and their characterization and expansion in vitro are vital processes. Perhaps the most exciting potential of cell therapy is our ability to manipulate cells by modifying their physiological properties before returning them to a patient. Cell therapy is evolving quickly, drawing on cell biology, molecular biology, virology, immunology and cell quantification techniques and also on biomedical engineering.These advances require well defined, reproducible and firmly established laboratory methods for investigating HSCs. The hematopoietic system comprises a concentrated series of stem and transit progenitor cell compartments of progressively restricted potentiality and proliferative capacity. In this setting, Hematopoietic Stem Cell Protocols, edited by Christopher A. Klug and Craig T. Jordan, is an essential handbook for novice and experienced investigators alike, which gives a wide variety of step-by-step instructions for the study of mouse and human HSCs of both embryonic and adult origin.The 20 chapters present techniques for stem cell analysis, some of which have become standardized over the past few years. Many chapters contain contributions by the researchers who first developed and introduced the procedures. Each chapter begins with a short general introduction referring to most of the main publications in the field, then the major part precisely reports the materials and methods, giving technical laboratory details or even ‘home-made cooking secrets’. Methods are described in the following areas: aorta–gonad–mesonephros and yolk sac HSCs; isolation of mouse HSCs; flow cytometric analysis and immunoselection of HSCs; purification of human HSCs by flow cytometry; cycling and turnover of HSC; cell-cycle analysis of primitive stem cells; hematopoietic colony-forming cells; long-term culture-initiating cell (LTC-IC) assays for human and murine cells; assays for cobblestone area-forming cells; colony forming unit on the spleen (CFU-S) assays; T-cell progenitor activity and competitive repopulation assays for HSCs in the NOD and SCID models. Protocols for stem cell expansion and the hematopoietic maturation of ES cells are included, as are detailed methods for the genetic modification of stem cells, for example, retroviral infection of murine HSCs, retroviral transduction of purified HSCs, retroviral-mediated transduction and HIV-based vectors. The last section deals with gene expression in HSCs and the use of two-dimensional gene-expression fingerprinting.This is a comprehensive compendium of practical techniques, a state-of-the-art handbook. It will be a useful tool for teams involved in either basic hematopoiesis research or progenitoror stem cell quantification for cell therapy protocols in the context of patient care. Thanks to a multidisciplinary approach, from fundamental to practical aspects, this book reports advances made in the field of hematopoiesis, which could subsequently be applied for diagnostic and therapeutic purposes.

Ampoule tests are commonly used as an index of the cryopreservation efficiency of marrow stem cells in bags. We have studied the recovery of hematopoietic progenitor cells (CFU-GM, BFUe) in 52 ampoules and compared it to the recovery in 83 standard bags. Our data showed significantly deficient CFU-GM and BFUe recoveries (respectively 47 +/- 31% and 31 +/- 30%) in ampoules when compared to bags (respectively 72 +/- 22% and 64 +/- 19%; P less than 0.001). Moreover, a good progenitor cell recovery (greater than or equal to 50%) was observed in only 46% of frozen ampoules versus 100% observed in frozen bags (P less than 0.05). We were able to relate this nonoptimal recovery to an excessively rapid freezing rate of -9 degrees C/min following the release of fusion heat which occurred in ampoules, while the freezing rate was constantly maintained at -2 degrees C/min in the corresponding bags. We therefore conclude that the cooling conditions have to be carefully controlled to ensure that the bags and ampoules are both cooled under the same conditions. Otherwise, ampoules would not be a reliable index of the true progenitor cells' cryopreservation efficiency in bags.

Prolonged in vitro maintenance of human bone marrow progenitor cells was achieved using a serum-free (SF) liquid culture system. Culture medium was based on Iscove's medium supplemented with bovine serum albumin, human transferrin, bovine insulin, soybean lecithin, cholesterol, hydrocortisone and alpha-thioglycerol. Under these standardized culture conditions, CFU-GM were maintained for up to 4 weeks, as is the case when using conventional serum-dependent medium. Erythropoiesis exhibited a slower decline than that found using serum containing medium. Development of normal haematopoiesis was effective in spite of poor stromal cell development--a confluent adherent layer as classically described in serum conditions was never achieved. Our newly defined system provides a reliable technique for studying human haematopoietic stem cell proliferation and differentiation in vitro; it allows for rational utilization of currently available purified recombinant growth factors. It may be a promising tool in the clinical use of cultured haematopoietic stem cells.

The in vitro treatment of leukemic bone marrows, collected during complete remission, aims at eliminating residual blast cells prior to freezing and preservation, while sparing normal hematopoietic stem cells. We report our experience on the activity of ASTA Z 7557 on human leukemic (CFU-L) and normal hematopoietic stem cells. The sensitivity of human leukemic and normal progenitor cells (CFU-c), detected in semi-solid media cultures, is similar. However, pre-CFUc progenitors detected in long term marrow cultures are much less sensitive to ASTA Z 7557. Therefore autologous bone marrow transplantation can successfully be done with pretreated marrows containing 5 +/- 5% residual CFUc. The wide range of stem cells sensitivity to ASTA Z 7557 justify the predetermination of the optimal dose of drug for incubation prior to marrow collection for each individual patient. Our preliminary clinical experience is exposed.

Blood nucleated cells collected by leukapheresis and spleen cell suspension from patients with myelofibrosis with myeloid metaplasia (MMM) were studied for their haematopoietic capacity. Using committed progenitor cell assays (CFU-GM, BFU-e) and a one-stage long-term liquid stem cell system, we have shown: (1) a preferential expansion of the circulating committed progenitor cell pool above the more primitive stem cell compartment; (2) the absence of any development of a stromal adherent layer in long-term cultures of peripheral blood nucleated cells suggesting the self-sustaining capacity of the circulating primitive stem cells; (3) that the spleen is only a production site of committed progenitor cells but does not generate primitive stem cells; (4) the presence, in the spleen, of stromal progenitor cells. We conclude that the peripheral blood primitive stem cells in patients with MMM are not of splenic origin.

Autologous bone marrow transplantation (ABMT) in chronic granulocytic leukemia (CGL) aims at reversing the acute or acceleration phases by injection of stem cells collected during the chronic phase. This study was designed to explain an unusual rate of delayed engraftment (50%) in our experience of ABMT in CGL patients. We investigated all the factors possibly responsible for abnormal perpetuation of aplasia following infusion of cryopreserved marrow stem cells. The study of CFU-gm recovery in 41 bags of frozen marrow from 25 patients revealed an overall deficiency with a mean CFU-gm recovery of 55 +/- 38% in CGL patients versus 73 +/- 15% in the control group (p less than 0.001). Our data also showed an inverse linear relation (r = -0.40, p less than 0.05) between CFU-gm concentration and recovery after freezing. A good CFU-gm recovery (greater than or equal to = 50%) was observed in 70% of cases when the concentration was less than 3700 CFU-gm/ml as compared to 30% of cases when the concentration was over 3700 CFU-gm/ml (p less than 0.001). The lack of improvement by diluting rich CFU-gm marrows to reduce CFU-gm concentration/ml, as well as the absence of relationship between CFU-gm recovery after freezing and nucleated cells concentration, suggest a particular fragility of CGL stem cells to freezing, probably related to their excessive amplification. At the present time, we strongly recommend that the highest possible dose of progenitor cells be cryopreserved, preferably at a low concentration, in patients with CGL, and particular attention devoted to the freezing procedure in each individual patient, with numerous appropriate efficiency tests.

The present study was conducted to establish defined culture conditions for ex vivo expansion of normal human bone marrow cells. We investigated the role of three experimental expansion parameters: the cell concentration in the initial culture medium, the role of animal serum, human plasma and serum-free substitute, and the expansion potential of mononucleated cells (MNC) versus CD34+ cells. Cells were cultured in suspension with stem cell factor (SCF), IL3, IL6 and Erythropoietin (Epo) for 10 days. 1) Reducing the cell concentration from 3 × 104 to 1.5 × 103/ml increased total cell expansion almost 20 fold, progenitor expansion more than 3 fold, and the maintenance of long term culture-initiating cells (LTC-IC). 2) In medium containing a serum-free substitute, total and CD34+ cell expansion was 3 times greater than in medium containing 1-10% human AB plasma or 25% animal serum. 3) The expansion potential of selected CD34+ cells was significantly greater than that of the total MNC population. However, taking into account the cell loss due to CD34+ selection, the overall results for quantitative expansion in relation to the initial number of MNCS favor the use of non-selected MNCS. 4) SCF+IL3+IL6 was clearly the best combination of early cytokines for LTC-IC maintenance, with or without lineage-restricted cytokines, whereas the presence of IL1s in any combination augmented the decrease in LTC-IC. Addition of G-CSF to the medium resulted in 1 log increase in total cell expansion and a 2-fold increase in CFU-GM expansion. Addition of Epo always induced a dramatic proliferation of erythroid cells (up to 2000 fold) as well as of CFU-GM (up to 4 fold), without exhausting the LTC-IC pool. We concluded that the expansion of hemopoietic cells for clinical purposes needs establishment of controlled, reproducible and reliable culture conditions.

The sensitivity of human myeloblastic leukemic (CFU-L) and normal hemopoietic stem cells (CFU-GM and BFU-e) to Asta Z 7557 (INN Mafosfamide) was studied with regard to autologous bone marrow transplantation (ABMT) with cleansed marrow for consolidation therapy in adult patients with acute leukemia (AL) in remission. Establishment of the dose-response curves for CFU-GM (n = 37), BFUe (n = 11), and myeloblastic CFU-L (n = 9) demonstrated a wide range of sensitivity from patient to patient for all three progenitors. Whereas CFU-L, CFU- GM, and BFU-e grown in semisolid cultures disclosed similar sensitivities to Asta Z 7557, long-term culture (LTC) studies (n = 41) indicated a higher resistance of early progenitors. In an effort to achieve a maximum tumor cell kill and yet spare a sufficient amount of normal stem cells to ensure consistent engraftment, we defined the optimal dose for marrow cleansing as the dose sparing 5% CFU-GM (LD95). This dose was established from a preincubation test (PIT) realized on a 10-mL marrow aspirate taken 15 days before marrow collection in each individual patient. Twenty-four adult patients while in remission of AL (20 in complete remission, four in partial remission) were consolidated by cyclophosphamide 60 mg/kg X 2 and total body irradiation at 10 Gy followed by ABMT with marrow cleansed by Asta Z 7557 according to the specification described above. Patients were divided in two groups: group 1, unfavorable prognosis (11 patients); group 2, standard prognosis [13 patients in first complete remission (CR)]. All patients engrafted on leukocytes (median day for recovery to 10(9)/L: day 30), patients with ALL recovered faster than patients with ANL (median day 19 v 34). Similarly, recovery of platelets to 50.10(9)/L occurred sooner in patients with ALL (median day 67, range day 23 through 90) whereas three patients with acute nonlymphoblastic leukemia (ANLL) in group 2 had to be supported with platelet transfusions for more than one year. In group 1, six patients had recurrent tumor within six months; three patients died from toxicity with no evidence of tumor. Two patients are still disease-free with a short follow-up (nine and ten months). In group 2, two patients died from toxicity with no evidence of leukemia three and 16 months post-ABMT. One patient with a M5 ANLL and one patient with ALL relapsed at six and 15 months, respectively. Nine patients have remained in CR or are disease-free with a median follow-up of 22 months.(ABSTRACT TRUNCATED AT 400 WORDS)