Contribution à l’étude de l’apoptose par la cytométrie en flux des cellules souches hématopoïétiques CD34+ avant et après le processus de congélation

Apoptosis represents a particular form of programmed cell death which appears in all the damaged cells and potentially hazardous. It plays a crucial role in the development of multicellular organisms by assuring and maintaining the cellular homeostasis. Thus, apoptosis intervenes not only in the normal process of organisms' development but also in immune defence and in cancerous cells detection. Indeed, any blockage in the program of the apoptotic machinery would be responsible of some neurodegenerative and auto-immune diseases and could play a crucial role in different steps of carcinogenesis. Some researchers were very interested in studying apoptosis in hematopoietic stem cells CD34+ which could be intended to be reinfused to patients suffering from malignant diseases. They have noted that kinetic study of apoptosis of the hematopoietic stem cells CD34+ after the process of cryoconservation is also necessary. Such study permits to quantify the real and exact number of the viable hematopoietic stem cells CD34+ and therefore to eliminate such risk which would be associated with the reinfusion of apoptotic cells to patients. In this paper, we describe our contribution to hematopoietic stem cells CD34+ study by flow cytometry before and after cryopreservation by using annexin V as a specific probe allowing detection of phosphatidyl serine, one of the major features of apoptosis. But, we have noted a pronounced induction of apoptosis in peripheral mobilized blood compared to cytapheresis (after cryopreservation: 29.79% of apoptotic HSC CD34+ in peripheral mobilized blood but only 11.67% apoptotic HSC CD34+ in cytapheresis). Besides, we have noticed that hematopoietic stem cells CD34+ have had a statute of viability better than other mononuclear cells. These results put in value the reliability, the simplicity and the efficiency of flow cytometry for the analysis of apoptosis in hematopoietic stem cells CD34+ by following the intensity of fluorescence of annexin V.     

A novel method of bone marrow transplantation (BMT) for intractable autoimmune diseases

We have previously proposed that autoimmune diseases are hemopoietic stem cell (HSC) disorders. In this review article, we provide evidence that most age-associated diseases such as osteoporosis are mesenchymal stem cell (MSC) disorders and, based on this evidence, we propose a new concept of "stem cell disorders (SCDs)", including HSC and MSC disorders. To treat SCDs, we have recently developed a new strategy (intra-bone marrow-bone marrow transplantation: IBM-BMT) for replacing the abnormal stem cells of recipients with donor-derived normal stem cells (both HSCs and MSCs). We here show that this strategy not only can be used to treat SCDs but is also applicable to organ transplantation, since IBM-BMT can induce tolerance (full chimerism) without the need for immunosuppressants even when radiation doses as the conditioning regimen of BMT are reduced to less than 5.0 Gy x 2, which is equivalent to one shot of 8 Gy (a sublethal dose). We believe that this strategy heralds a revolution in the field of transplantation (BMT and organ transplantation) and regeneration therapy.     

Delineation of the human hematolymphoid system: potential applications of defined cell populations in cellular therapy

Summary: Hematopoietic stem cells (HSC) have the capacity to reconstitute ail the blood cells in the body HSC are rare, representing on average 0.0 5% of the mononuclear cells present in healthy human bone marrow. Due to their capacity for self–renewal and their pluripotent, long–term reconstituting potential. HSC are considered ideal for transplantation to reconstitute the hematopoietic system after treatment for various hematologic disorders or as a target for the delivery of therapeutic genes. Human HSC also have potential applications in restoring the immune system in autoimmune diseases and in the induction of tolerance for allogeneic solid organ transplantation. With the increased interest in human HSC for clinical applications, technology for the isolation of candidate HSC and knowledge of human hematopoiesis have been growing rapidly. In this article, we discuss the functional characterization of a human CD34⁺ Thy-1⁺ HSC population which is essentially free of residual disease, our efforts to generate alternate monoclonal antibodies for the isolation of clinically useful stem or progenitor cell populations, and the identification of a novel lymphoid progenitor as part of an exploration towards defining progenitors with potential application as adjuncts to HSC–based cellular therapy.     

造血干细胞的可塑性及临床应用

干细胞是一类具有自我复制和多分化潜能的细胞 ,根据其发育阶段不同 ,分为胚胎干细胞和成体干细胞。胚胎干细胞具有多分化潜能 ;而成体干细胞的分化潜能则较为局限 ,只能定向分化为特化细胞。但新近的研究表明 ,成体干细胞在一定条件下可分化为与其所在组织不同的其他组织类型细胞 ,这种干细胞的可塑性被称为成体干细胞的横向分化(ｔｒａｎｓｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ)。其中造血干细胞 (ｈｅｍａｔｏｐｏｉｅｔｉｃｓｔｅｍｃｅｌｌｓ,ＨＳＣ)作为人们认识最深入 ,其临床应用也最为广泛的一类成体干细胞 ,目前已成为成体干细胞可塑性…     

Préparation de produits de thérapie cellulaire: apport des systèmes clos

A phase I clinical trial is being currently performed in our institution, aiming at evaluating the feasibility and toxicity related to the administration of Herpes Simplex-thymidine kinase gene-expressing human primary T lymphocytes following allogeneic hematopoietic stem cell transplantation. The need for safe and standardized preparation conditions for gene-modified cells is crucial We describe the closed culture system used in the current trial for ex vivo retroviral-mediated gene transfer and transduced cell selection. Cell handling is performed in closed systems using sampling and transfer pack bags, culture bags and a sterile connection device which avoids opening the culture system. This closed system allows safe and reproducible ex vivo preparation of gene-modified primary T-lymphocytes for clinical use.     

An Evolving Model of Hematopoietic Stem Cell Functional Identity

Easily accessed via venipuncture, blood has been an object of study for centuries. Direct sampling of the hematopoietic tissues in the bone marrow also presents a rather low bar for biopsy acquisition from living donors, especially when compared to other systems in the body such as the heart or brain. This relatively straight-forward ability to obtain cells from the primary anatomical locations of blood cell genesis and differentiation combines with a reliable transplantation assay and well-described surface markers to make the hematopoietic stem cell (HSC) the best understood of all tissue stem cells. HSC biology has been extensively though incompletely investigated over the years. The field continually refreshes itself as new findings require us to reevaluate our understanding of hematopoiesis. After providing a brief overview of the hematopoietic system in general, this review will touch on recent findings in three areas: (1) the niche, (2) HSC migration, and (3) challenges to the "classical" model of hematopoietic ontogeny.     

Hematopoietic stem and progenitor cell trafficking

Migration of hematopoietic stem cells (HSCs) is essential during embryonic development and throughout adult life. During embryogenesis, trafficking of HSCs is responsible for the sequential colonization of different hematopoietic organs by blood-producing cells. In adulthood, circulation of HSCs maintains homeostasis of the hematopoietic system and participates in innate immune responses. HSC trafficking is also crucial in clinical settings such as bone marrow (BM) and stem cell transplantation. This review provides an overview of the molecular and cellular signals that control and fine-tune trafficking of HSCs and hematopoietic progenitor cells in embryogenesis and during postnatal life. We also discuss the potential clinical utility of therapeutic approaches to modulate HSC trafficking in patients.     

Bone marrow tissue engineering

The creation of mixed hematopoietic chimerism has become an important clinical strategy for tolerance induction for cellular and organ transplantation, and for the treatment of numerous hematopoietic diseases. Clinical success has been limited however, by host immune response and by competition from host hematopoiesis. Recent data suggests that limited donor stem cell engraftment after minimally myeloablative hematopoietic stem cell (HSC) transplantation may in part be due to MHC associated microenvironmental mismatch resulting in a competitive disadvantage for donor HSC. A strategy to overcome this barrier to stable mixed hematopoietic chimerism would involve concurrent transplantation of a donor bone marrow microenvironment. To test this possibility, we set out to develop a method to tissue engineer a bone marrow microenvironment. One to two murine femurs were mechanically crushed to a fine suspension and were combined in vitro with various delivery vehicles. These constructs were transplanted into syngeneic animals in locations that are known to support transplantation of other tissues. Although bone formation was observed with several conditions, bone marrow formation was noted only within the small bowel mesentery when type I collagen was used as the delivery vehicle. No bone marrow formed when the vehicle was changed to polyglycolic acid or type IV collagen. We have demonstrated that the small bowel mesentery can support bone marrow formation under specific in vivo conditions. Future work will focus on strategies for transplantation of an engineered donor bone marrow environment to facilitate creation of allogeneic mixed hematopoietic chimerism.     

The normal flora may contribute to the quantitative preponderance of myeloid cells under physiological conditions

Under physiological conditions, the innate immune cells derived from myeloid lineage absolutely outnumber the lymphoid cells. At present, two theories are attributed to the maintenance of haemopoiesis: the asymmetric cell division and the bone marrow hematopoietic microenvironment or "niche". However, the former only explains the self-renewal of haemopoietic stem cell (HSC) and the start of haemopoietic differentiation but fails to address the inducers of cell fate decisions; the latter has to admit that the hematopoietic cytokines, despite their significance in the maintenance of haemopoiesis, have no specific effect on lineage commitment. Given these flaws, the advantageous mechanism of myeloid haemopoiesis has not yet been uncovered in the current theories. The discoveries that bacterial components (lipopolysaccharide, LPS) and intestinal decontamination affect the mobilization of HSC trigger the interest in normal flora, which together with their components may have an effect on haemopoiesis. In the experiments in dogs and mice, researchers documented that the generation of myeloid cells has undergone changes in the bone marrow and periphery when antibiotics are used to regulate the normal intestinal flora and the concentration of its components. However, the same changes are not involved in lymphoid cells. Therefore, we hypothesize that in human body normal flora and its components are a driving force to maintain myeloid haemopoiesis under physiological conditions. To account for the selectiveness in haemopoiesis, these facts should be taken into consideration, such as HSC and mesenchymal stem cells (MSC) functionally expressed pattern recognition receptors (PRR), and both of them can self-migrate or be recruited by normal flora or its components into periphery. Dynamically monitoring the myeloid haemopoiesis may provide an important complementary program that precludes the abuse of antibiotics, which prevents diseases triggered by the imbalance of normal flora. Meanwhile, the regulation of normal flora and the use of purified microecological modulator may serve as valuable auxiliary treatments to mobilize HSC prior to the HSC transplantation as well as to promote hematopoietic recovery after transplantation or chemotherapy in the blood diseases.     

Do We Have a Workable Clinical Protocol for Differentiating Lympho‐Hematopoietic Stem Cells from the Source of Embryonic Stem Cells and Induced Pluripotent Stem Cells in Culture?

In recent years, many researchers are focusing on deriving lympho‐hematopoietic stem cells (L‐HSC) from human embryonic stem cells (ESC) and/or induced pluripotent stem cells (iPSC) in culture as alternative sources for transplantation. Two protocols are available for research purposes: mouse stroma cell line coculture system and embryoid bodies (EBs) suspension culture system. However, due to the lack of human stroma cell line, which could support the derivation of L‐HSC in culture, the generation of therapeutic lympho‐hematopoietic cells for clinical purpose can only be achieved using EBs suspension culture system. In this short communication/review, the results of EBs suspension culture system using mouse and human ESC/iPSC are summarized and the potential clinical application is discussed.     

Asymmetry of stem cell fate and the potential impact of the niche

Asymmetric cell division is a common concept to explain the capability of stem cells to simultaneously produce a continuous output of differentiated cells and to maintain their own population of undifferentiated cells. Whereas for some stem cell systems, an asymmetry in the division process has explicitly been demonstrated, no evidence for such a functional asymmetry has been shown for hematopoietic stem cells (HSC) so far. This raises the question regarding whether asymmetry of cell division is a prerequisite to explain obvious heterogeneity in the cellular fate of HSC. Through the application of a mathematical model based on self-organizing principles, we demonstrate that the assumption of asymmetric stem cell division is not necessary to provide a consistent account for experimentally observed asymmetries in the development of HSC. Our simulation results show that asymmetric cell fate can alternatively be explained by a reversible expression of functional stem cell potentials, controlled by changing cell-cell and cell-microenvironment interactions. The proposed view on stem cell organization is pointing to the potential role of stem cell niches as specific signaling environments, which induce developmental asymmetries and therefore, generate cell fate heterogeneity. The self-organizing concept is fully consistent with the functional definition of tissue stem cells. It naturally includes plasticity phenomena without contradicting a hierarchical appearance of the stem cell population. The concept implies that stem cell fate is only predictable in a probabilistic sense and that retrospective categorization of stem cell potential, based on individual cellular fates, provides an incomplete picture.     

Notch signaling and the emergence of hematopoietic stem cells

The hematopoietic stem cell (HSC) is able to give rise to all blood cell lineages in vertebrates. HSCs are generated in the early embryo after two precedent waves of primitive hematopoiesis. Canonical Notch signaling is at the center of the complex mechanism that controls the development of the definitive HSC. The successful in vitro generation of hematopoietic cells from pluripotent stem cells with the capacity for multilineage hematopoietic reconstitution after transplantation requires the recapitulation of the most important process that takes place in the hemogenic endothelium during definitive hematopoiesis, that is the endothelial-to-hematopoietic transition (EHT). To meet this challenge, it is necessary to thoroughly understand the molecular mechanisms that modulate Notch signaling during the HSC differentiation process considering different temporal and spatial dimensions. In recent years, there have been important advances in this field. Here, we review relevant contributions describing different genes, factors, environmental cues, and signaling cascades that regulate the EHT through Notch interactions at multiple levels. The evolutionary conservation of the hematopoietic program has made possible the use of diverse model systems. We describe the contributions of the zebrafish model and the most relevant ones from transgenic mouse studies and from in vitro differentiated pluripotent cells.     

Space-time considerations for hematopoietic stem cell transplantation

The mammalian blood system contains a multitude of distinct mature cell lineages adapted to serving diverse functional roles. Mutations that abrogate the development or function of one or more of these lineages can lead to profound adverse consequences, such as immunodeficiency, autoimmunity, or anemia. Replacement of hematopoietic stem cells (HSC) that carry such mutations with HSC from a healthy donor can reverse such disorders, but because the risks associated with the procedure are often more serious than the blood disorders themselves, bone marrow transplantation is generally not used to treat a number of relatively common inherited blood diseases. Aside from a number of other problems, risks associated with cytoreductive treatments that create "space" for donor HSC, and the slow kinetics with which immune competence is restored following transplantation hamper progress. This review will focus on how recent studies using experimental model systems may direct future efforts to implement routine use of HSC transplantation to cure inherited blood disorders.     

Modalités de conservation et de destruction des produits cellulaires cryopréservés : recommandations de la SFGM-TC

Thousands of autologous and at less extent allogeneic hematopoietic stem cells (HSC) bags are cryopreserved in France. The majority of autologous HSC grafts are used within a year after collection. However, many bags are still unused and cryopreserved for many years. In France and on a European scale, the ever-growing number of cryopreserved bags represents a real economic health concern. Indeed, the cost of storage is about 100 € per bag and per year. In addition, quality and therapeutic value of these long-term cryopreserved grafts needs to be evaluated. In the attempt to harmonize clinical practices between different French transplantation centers, the French Society of Bone Marrow Transplantation and Cell Therapies (SFGM-TC) set up its fourth annual series of workshops which brought together practitioners from its member centers across France. These workshops took place in September 2013 in Lille. In this article, we addressed the issue of the destruction of long-term cryopreserved grafts be them autologous or allogeneic and provide recommendations regarding their destruction.     

Culture de cellules à visée transfusionnelle : le cas des globules rouges

One has been trying for several years to find a substitute for red blood cells (RBC). The development of chemical or natural molecules to replace hemoglobin has nevertheless proved difficult and artificial blood is still unattainable. We have described a methodology permitting the massive ex vivo production of mature human RBC having all the characteristics of native adult RBC from hematopoietic stem cells (HSC) of diverse origins: blood, bone marrow or cord blood. This protocol allows both massive expansion of the HSC/progenitors and their complete differentiation to the stage of perfectly functional mature RBC. The levels of amplification obtained (10⁵ to 2 × 10⁵) are compatible with an eventual transfusion application. We discuss here the state of the art of this new concept and evoke the obstacles to be overcome to pass from the laboratory model to clinical practice. This concept of “cultured RBC” opens up potentially considerable therapeutic perspectives in the field of blood transfusion.     

造血干细胞移植治疗恶性血液病的研究与分析

背景：近年来,随着血液学及相关领域基础和临床研究的不断发展,造血干细胞移植已成为治疗恶性血液病的有效手段,甚至是治愈某些血液病的惟一方法。 目的：对造血干细胞移植治疗恶性血液病SCI数据库文献资料进行多层次对比分析。 方法：通过计算机检索SCI数据库2001/2010有关造血干细胞移植治疗恶性血液病的文献,检索词为“造血干细胞(hematopoietic stem cell),移植(transplantation),急性髓性白血病(acute myelogenous leukemia),非霍奇金淋巴瘤(non-hodgkin lymphoma),慢性髓细胞白血病(chronic myelocytic leukemia)”,分析结果以文字和图表的形式进行统计和计量学分析。 结果与结论：SCI数据库2001/2010收录造血干细胞移植治疗恶性血液病相关文献中美国在该领域文献产出量多于其他国家,对该领域研究有重要贡献,发表文献较多的机构集中在美国Fred Hutchinson癌症研究中心。10年来文献数量呈总体上升趋势。     

Expression and function of homing-essential molecules and enhanced in vivo homing ability of human peripheral blood-derived hematopoietic progenitor cells after stimulation with stem cell factor

Hematopoietic stem cell (HSC) homing from blood to bone marrow is a multistep process involving rolling, extravasation, migration, and finally adhesion in the correct microenvironment. With view to the hematopoietic recovery after clinical stem cell transplantation, we investigated the effect of stem cell factor (SCF) on the expression and the adhesive function of the alpha4beta1 and alpha5beta1 integrins very-late antigen (VLA)-4 and VLA-5 on peripheral blood-derived hematopoietic progenitor cells. After SCF stimulation, the expression of VLA-4 and VLA-5 on CD34+/c-kit+ cells obtained from healthy donors increased from 54% to 90% and from 3% to 82%, respectively. For patient-derived cells, the increase was 67% to 90% and 12% to 46%. The proportion of mononuclear cells adhering to the fibronectin fragment CH296 increased by stimulation with SCF from 14% to 23%. Accordingly, functional studies showed an approximate 30% increase of adherent long-term culture-initiating cell. The improvement of the homing abilities of SCF-stimulated HSC was confirmed by transplantation into sublethally irradiated nonobese diabetic-scid/scid mice. Six weeks after the transplantation, in eight of eight animals receiving human HSC with the addition of SCF, a profound multilineage hematopoietic engraftment was detected, whereas in the control group receiving only HSC, none of eight animals engrafted. Our data provide the first in vivo evidence that stimulation with cytokines improves the homing ability of transplanted human hematopoietic progenitor cells.     

Antioxidant intervention: a new method for improving hematopoietic reconstitution capacity of peripheral blood stem cells

In peripheral blood stem cell transplantation, bone marrow hematopoietic stem cells (BM HSCs) are collected as they are released from the hypoxic bone marrow, then infused into peripheral blood with higher oxygen concentration after mobilization. In some cases, in vitro amplification culture under normal oxygen may be required, and homing into the hypoxic bone marrow is further carried out after intravenous re-infusion, thereby resulting in constant changes in the reactive oxygen species (ROS). The high-level ROS can damage the hematopoietic reconstitution capacity of HSCs. Thus, the application of antioxidant intervention in the in vivo mobilization of BM HSC and the in vitro culture process of peripheral blood stem cells may be effective against the negative effects of ROS on BM HSC. Antioxidant intervention may also better protect the hematopoietic reconstitution capacity of HSCs, as well as improve the success rate of transplantation.     

Cotransplantation of cord blood hematopoietic stem cells and culture-expanded and GM-CSF-/SCF-transfected mesenchymal stem cells in SCID mice

Mesenchymal stem cells (MSC) are multipotent in nature and believed to facilitate the engraftment of hematopoietic stem cells (HSC) when transplanted simultaneously in animal studies and even in human trials. In this study, we transfected culture-expanded MSC with granulocyte macrophage-colony stimulating factor (GMCSF) and stem cell factor (SCF) cytokine genes and then cotransplanted with mononuclear cells (MNC) to further promote HSC engraftment. MNC were harvested from cord blood and seeded in long-term culture for ex vivo MSC expansion. A total of 1 x 10(7) MNC plus MSC/microL were introduced to the tail vein of nonobese diabetic/severe combined immunodeficiency mice. After 6-8 weeks later, homing and engraftment of human cells were determined by flow cytometry and fluorescence in situ hybridization studies. The total nucleated cell count and the engraftment of CD45+/CD34+ cells and XX or XY positive human cells were significantly increased in cotransplanted mice and even higher with the cytokine gene-transfected MSC (GM-CSF>SCF, p<0.05) than in transplantation of MNC alone. These results suggest that MSC transfected with hematopoietic growth factor genes are capable of enhancing the hematopoietic engraftment. Delivering genes involved in homing and cell adhesions, CXCR4 or VLA, would further increase the efficiency of stem cell transplantation in the future.     

Hematopoietic stem cell therapy for type 1 diabetes: induction of tolerance and islet cell neogenesis

Diabetes is a chronic disease with significant morbidity and mortality. Pancreas or islet cell transplantation is limited by a shortage of donors and chronic immune suppression to prevent allograft rejection. Consequently, interest exists in islet cell neogenesis from embryonic or mesenchymal stem cell as a possible cure for diabetes. However, unless tolerance to islet cells is re-established, diabetes treated by islet cell transplantation would remain a chronic disease secondary to immune suppression related morbidity. If islet cell tolerance could be re-induced, a major clinical hurdle to curing diabetes by islet cell neogenesis may be overcome. Recent studies suggest that adult hematopoietic stem cells (HSC) can reintroduce tolerance to auto-antigens. It is possible that HSC may also be able to switch lineage and, therefore, be a convenient source of stem cells for both inducing tolerance and islet cell regeneration.