Severe damage of human megakaryocytopoiesis and thrombopoiesis by heavy-ion beam radiation

Heavy ions have a unique efficacy for tumor control in radiotherapy. To clarify the effects of heavy-ion beams on hematopoietic stem/progenitor cells, the effects of carbon-ion beams on megakaryocytopoiesis and thrombopoiesis in CD34(+) cells derived from human placental and umbilical cord blood were investigated. The cells were exposed to carbon-ion beams (LET = 50 keV/microm) and then were treated with thrombopoietin (TPO) alone or TPO plus other cytokines. Megakaryocytic progenitor cells, such as megakaryocyte colony-forming units (CFU-Meg), were far more sensitive to carbon-ion beams than to X rays, and no restoration of carbon-ion beam-irradiated CFU-Meg by treatment with any cytokine combination was observed. However, total cell expansion in liquid culture was not different after either carbon-ion beam or X irradiation of CD34(+) cells. The activation of gamma-H2AX, a marker of DNA double strand-breaks (DSBs), was promoted by the cytokine treatment in X-irradiated CD34(+) cells but not in carbon-ion-irradiated cells. These results showed that carbon-ion beams inflicted severe damage on megakaryocytopoiesis and thrombopoiesis and that a better combination of cytokines and other agents may be needed to stimulate the recovery of hematopoietic cells and repair this damage.     

Placental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells support hematopoietic recovery of X-irradiated human CD34+ cells

AimsThe potential of human mesenchymal stem cell-like stroma prepared from placental/umbilical cord blood for hematopoietic regeneration by X-irradiated hematopoietic stem cells is herein assessed.Main methodsPlacental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells were applied to a regenerative ex vivo expansion of X-irradiated human CD34⁺ cells in a serum-free liquid culture supplemented with a combination of interleukine-3 plus stem cell factor plus thrombopoietin.Key findingsThe total number of cells and of lineage-committed myeloid hematopoietic progenitor cells generated in the co-culture of both non-irradiated and X-irradiated cells with stromal cells was significantly higher than those in the stroma-free culture. In addition, the number of CD34⁺ cells and CD34⁺/CD38^? cells, immature hematopoietic stem/progenitor cells also increased more than the stroma-free culture. The stromal cells produced various types of cytokines, although there was little difference between the co-cultures of non-irradiated and X-irradiated cells with stromal cells. Furthermore, when X-irradiated cells came in contact with stromal cells for 16 h before cytokine stimulation, a similar degree of hematopoiesis was observed, thus suggesting the critical role of cell-to-cell interaction.SignificanceThe present results showed the potential efficacy of human mesenchymal stem cell-like stroma for hematopoietic regeneration from irradiated hematopoietic stem/progenitor cells.     

Efficient enucleation of erythroblasts differentiated in vitro from hematopoietic stem and progenitor cells

Erythroblast enucleation is thought to be largely dependent on signals mediated by other cells, such as macrophages. In an attempt to improve the in vitro production of red blood cells (RBCs) from immature hematopoietic progenitor cells, we have developed a method to produce enucleated RBCs efficiently in the absence of feeder cells. Our method may represent an efficient way to produce transfusable RBCs on a large scale from hematopoietic progenitors.     

胚胎干细胞向血液干细胞定向分化的研究进展

骨髓移植是目前治疗恶性白血病以及遗传性血液病最有效的方法之一。但是HLA相匹配的骨髓捐献者严重短缺,骨髓造血干细胞（hematopoietic stem cells,HSCs）体外培养困难,在体外修复患者骨髓造血干细胞技术不成熟,这些都大大限制了骨髓移植在临床上的应用。多能性胚胎干细胞（embryonic stem cells,ESCs）具有自我更新能力,在合适的培养条件下分化形成各种血系细胞,是造血干细胞的另一来源。在过去的二十多年里,血发生的研究是干细胞生物学中最为活跃的领域之一。小鼠及人的胚胎干细胞方面的研究最近取得了重大进展。这篇综述总结了近年来从胚胎干细胞获得造血干细胞的成就,以及在安全和技术上的障碍。胚胎干细胞诱导生成可移植性血干细胞的研究能够使我们更好地了解正常和异常造血发生的机制,同时也为造血干细胞的临床应用提供理论和实验依据。     

Early fetal hematopoietic development from in vitro differentiated embryonic stem cells.

In this report we describe the efficient hematopoietic differentiation of embryonic stem (ES) cells in vitro. When cultured in semisolid medium two of five ES cell lines efficiently generated embryoid bodies (EBs) containing blood islands in which hematopoietic cells from all six myeloid lineages could be detected. Among a variety of growth factors tested, only erythropoietin significantly increased blood island formation. We directly demonstrate the presence of hematopoietic progenitors in the EBs by employing an in vitro precursor assay. Colony-forming cells (CFC) of all myeloid lineages as well as bi- and multipotent (CFC-MIX) progenitors were readily identified, and a detailed time-course analysis of their appearance was performed. Despite a high frequency of CFC-MIX in vitro, we did not observe any spleen colony-forming cells (CFU-S) in vivo. We conclude that hematopoietic differentiation of ES cells under these conditions reflects formation of the complete range of blood cells found in the yolk sac of the early fetus. Therefore this system provides a unique model in which to study the earliest events of hematopoietic development in vitro.     

Water permeability of human hematopoietic stem cells

It is currently impossible to isolate or identify human hematopoietic progenitor cells from the bone marrow, yet the biophysical properties of these cells are important for the development of techniques to isolate and preserve stem cells for transplantation. Osmotic permeability properties of human bone marrow stem cells were estimated from the kinetics of cell damage in a hypotonic solution measured using in vitro colony assays for multipotential (CFU-GEMM) and committed (BFU-E, CFU-GM) progenitor cells. Cells exposed to a hypotonic solution swell as a result of water influx, and the rate of change of volume is proportional to the hydraulic conductivity of the plasma membrane. Cell damage occurs when the cell volume exceeds the maximum tolerable volume, so the hydraulic conductivity can be estimated from the kinetics of cell damage. For all the progenitor cells studied, the mean value of the hydraulic conductivity was 0.283 micron3/micron2/min/atm at 20 degrees C, with an Arrhenius activation energy of 6.41 kcal/mole. No significant differences were observed in the osmotic properties of the various progenitor cells. These data were used to predict the osmotic responses of human bone marrow stem cells at subzero temperatures during freezing.     

Derivation of hematopoietic stem cells from murine embryonic stem cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny. Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability. Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types. The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol. HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders. However, many patients that might benefit from HSC therapy lack access to suitable donors. ESC could provide an alternative source of HSC for these patients. The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC. In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation. EBs are then dissociated and infected with retroviral HoxB4. Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days. During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100 s of millions of cells. These cells can then be used to rescue and reconstitute lethally irradiated mice.     

The human hematopoietic stem cell in vitro and in vivo.

A quantitative assay for a primitive human hematopoietic cell has been developed. The cell identified has been assigned the operational designation of long-term culture (LTC)-initiating cell based on its ability when cultured on supportive fibroblast monolayers to give rise to daughter cell(s) detectable by standard in vitro colony assays. Three lines of evidence support the view that the LTC-initiating cell assay may allow the relatively specific enumeration of totipotent cells with in vivo reconstituting potential. These involve the demonstration: (1) that conditions in analogous murine long-term cultures stimulate the extensive amplification (self-renewal) of some totipotent long-term repopulating cells, (2) that most of the LTC-initiating cells in normal human bone marrow are phenotypically different from most of the colony-forming cells present in the same cell suspensions in their possession of a number of characteristics specifically associated with transplantable stem cells; and (3) that cultured marrow cells from patients with chronic myeloid leukemia which, after maintenance under LTC conditions for 10 days contain some normal LTC-initiating cells but no detectable leukemic LTC-initiating cells, can after autografting reconstitute the hematopoietic system with normal cells.     

In vitro characterization of fetal hematopoietic stem cells: range and kinetics of cell production from individual stem cells

We have developed methods for detailed characterization of the proliferation kinetics and lineage potential of single human hematopoietic progenitor cells in an in vitro culture system. Fetal bone marrow CD34(hi)/lin(-) cells were cultured at one cell per well in the presence of c-kit ligand (KL), interleukin (IL)-3, IL-6, and leukemia inhibitory factor (LIF) on a murine stroma cell monolayer. Individual wells were scored for growth between 1 and 10 weeks of culture and analyzed by flow cytometry for lineage composition. A wide variation in time (1 to 8 weeks) was observed before initial cell division, even in the presence of cytokines promoting cell division in primitive progenitors. Eleven percent of the plated cells eventually produced a confluent culture well of approximately 20,000 progeny. Confluent wells were harvested and individually analyzed by flow cytometry for cell surface phenotype. Forty-eight percent of confluent wells contained primitive progenitors (CD34(+)lin(-)), 16% contained B-lymphoid cells (CD19(+) or CD10(+)), and 100% contained cells committed to the myelo-erythroid lineage (CD33(+)). CD34(+)/lin(-) cells from confluent wells were replated at one cell per well in secondary culture and the analysis repeated. One of 216 original single cells plated produced populations of B-lymphoid cells, myeloid cells, and primitive progenitors (CD34(+)/lin(-)) which persisted through two expansion cycles. We estimate that more than 36 million cells can be produced from a single cell under these culture conditions. A very small percentage of the CD34(hi)/lin(-) population (about 1%) was responsible for the majority of subsequent cell production. Our estimate of stem cell content in fetal bone marrow, defined by self-renewal as well as both B-lymphoid and myeloid differentiation from one cell, is approximately 1/13,000. This assay system provides direct in vitro measurements of the expected characteristics of hematopoietic stem cells (high proliferation potential, multilineage potential, self-renewal, and quiescence), and is therefore well suited to assessment of stem cell activity within various cell populations. (c) 1996 John Wiley & Sons, Inc.     

Hepatic regeneration from hematopoietic stem cells

In recent years, numerous investigators have reported novel cellular fates of multipotent stem or progenitor cells. In this review, we discuss the unexpected observations that hematopoietic stem cells can contribute to the hepatocyte lineage in humans and in rodent models of liver disease and regeneration. A key unresolved issue regarding hepatic regeneration from hematopoietic stem cells is whether the mechanism occurs through transdetermination, cell fusion, or other processes. A better understanding of the various stem or progenitor cells of the hepatic lineage may facilitate cellular transplantation approaches for the correction of hepatic function in patients with end-stage liver disease.     

CAPE promotes the expansion of human umbilical cord blood-derived hematopoietic stem and progenitor cells in vitro

Due to the low number of collectable stem cells from single umbilical cord blood(UCB)unit,their initial uses were limited to pediatric therapies.Clinical applications of UCB hematopoietic stem and progenitor cells(HSPCs)would become feasible if there were a culture method that can effectively expand HSPCs while maintaining their self-renewal capacity.In recent years,numerous attempts have been made to expand human UCB HSPCs in vitro.In this study,we report that caffeic acid phenethyl ester(CAPE),a small molecule from honeybee extract,can promote in vitro expansion of HSPCs.Treatment with CAPE increased the percentage of HSPCs in cultured mononuclear cells.Importantly,culture of CD34+HSPCs with CAPE resulted in a significant increase in total colony-forming units and high proliferative potential colony-forming units.Burst-forming unit-erythroid was the mostly affected colony type,which increased more than 3.7-fold in 1μg mL 1CAPE treatment group when compared to the controls.CAPE appears to induce HSPC expansion by upregulating the expression of SCF and HIF1-α.Our data suggest that CAPE may become a potent medium supplement for in vitro HSPC expansion.     

Emergence of hematopoietic stem cells in the human embryo

We have previously identified a novel site of hematopoietic cell production within the human embryo, which is localised in the ventral wall of the dorsal aorta and vitelline artery. Cells emerging in that territory between 27 and 40 days of gestation exhibit the expected phenotypic, molecular, and functional properties of hematopoietic stem cells and are the first multipotent, lympho-myeloid progenitors that appear in human ontogeny. We have next demonstrated that vascular endothelial cells sorted stringently, by flow cytometry, from the human yolk sac and embryonic aorta exhibit dramatic blood-forming potential in culture. These results suggest a filiation between vascular endothelium and hematopoietic cells in the course of early human ontogeny. More preliminary data indicate that a subpopulation of vascular endothelial cells in the bone marrow may retain this hematogenous potential until adult stages.     

Lymphocyte development from hematopoietic stem cells

The recent application of new techniques, such as multi-color cell sorting and the production of transgenic and gene-knockout mice, has contributed to a better understanding of lymphocyte development from hematopoietic stem cells. Now that we can purify progenitors at different maturational stages during lymphocyte development, the challenge is to understand the processes that govern each developmental stage transition.     

Endothelial cells from hematopoietic stem cells are functionally different from those of human umbilical vein

Hematopoietic stem cells have a remarkable plastic capacity, which allows them to differentiate into various cells, such as immune cells, nervous cells, muscle cells, bone and cartilaginous cells. The aim of this study was to show the capacity of stem cells to differentiate into endothelial cells, in culture, after addition of endothelial cells growth suplement (ECGS). We also compared the behavior of these cells with that of endothelial cells obtained from human umbilical vein (HUVEC). CD34+ cells obtained by immunomagnetic separation from human umbilical cord and placental blood were used. After 12-15 days of culture in a medium containing ECGS, the cells showed morphological changes characteristic to endothelial cells and immunocytochemical analysis revealed the presence of CD31 surface antigen and von Willebrand factor. The flow-cytometric analysis of endothelial cells adhesion molecules (ECAM) showed that endothelial cells derived from CD34+ cells expressed CD54/ICAM-1 9.65 ± 0.2% and CD106/VCAM 7.73±0.3%, values similar to those expressed by HUVECs. After TNF incubation, ECAM expression increased only in HUVECs. These data demonstrate that a fraction of circulating CD34+ cells may develop some endothelial cell characteristics when cultured with ECGS, but they are functionally different from HUVECs.     

Co-transplantation of human hematopoietic stem cells and human breast cancer cells in NSG mice

Humanized tumor mice (HTM) were generated by the co-transplantation of human hematopoietic stem cells and human breast cancer cells overexpressing HER2 into neonatal NOD-scid IL2Rγ^null (NSG) mice. These mice are characterized by the development of a human immune system in combination with human breast cancer growth. Due to concurrent transplantation into newborn mice, transfer of MHC-mismatched tumor cells resulted in solid coexistence and immune cell activation (CD4⁺ T cells, natural killer cells, and myeloid cells), but without evidence for rejection. Histological staining of the spleen of HTM revealed co-localization of human antigen-presenting cells together with human T and B cells allowing MHC-dependent interaction, and thereby the generation of T cell-dependent antibody production. Here, we investigated the capability of these mice to generate human tumor-specific antibodies and correlated immunoglobulin titers with tumor outgrowth. We found detectable IgM and also IgG amounts in the serum of HTM, which apparently controlled tumor development when IgG serum concentrations were above 10 µg/ml. Western blot analyses revealed that the tumor-specific antibodies generated in HTM did not recognize HER2/neu antigens, but different, possibly relevant antigens for breast cancer therapy. In conclusion, HTM offer a novel approach to generate complete human monoclonal antibodies that do not require further genetic manipulation (e. g., humanization) for a potential application in humans. In addition, efficacy and safety of the generated antibodies can be tested in the same mouse model under human-like conditions. This might be of particular interest for cancer subtypes with no currently available antibody therapy.