Autologous cell therapy as a new approach to treatment of radiation-induced bone marrow aplasia: preliminary study in a baboon model

The sparing of viable hematopoietic stem and progenitor cells located in underexposed bone marrow territories associated with the relative radioresistance of certain stem cell populations is the rationale for autologous cell therapy consisting of ex vivo expansion of residual cells after collection postirradiation. The feasibility of this treatment mainly depends on time constraints and hematopoietic cell threshold. We showed in this study that in the absence of early-acting mobilizing agent administration, subliminar amounts of CD34+ cells can be collected (1 x 10(6) CD34+ cells/100 mL bone marrow or for 1 L apheresis) from 6-Gy gamma globally irradiated baboons. Residual CD34+ cells were successfully expanded in serum-free medium in the presence of antiapoptotic cytokine combination (stem cell factor + FLT-3 ligand + thrombopoietin + interleukin 3, 50 ng/mL each, i.e., 4F): KCD34+ = x2.8 and x13.7 (n = 2). Moreover, we demonstrated the short-term neutrophil engraftment potential of a low-size mixed expanded graft (1.5 x 106 final CD34+cells/kg) issued from the coculture of unirradiated (20%) and 2.5-Gy in vitro irradiated (80%) CD34+ cells on an allogeneic stromal cell layer in the presence of 4F. Further preclinical research needs to be performed to clearly establish this therapeutic approach that could be optimized by the early administration of antiapoptotic cytokines.     

Mesenchymal stem cells support expansion of in vitro irradiated CD34(+) cells in the presence of SCF, FLT3 ligand, TPO and IL3: potential application to autologous cell therapy in accidentally irradiated victims

Ex vivo expansion of residual autologous hematopoietic stem and progenitor cells collected from victims soon after accidental irradiation (autologous cell therapy) may represent an additional or alternative approach to cytokine therapy or allogeneic transplantation. Peripheral blood CD34+ cells could be a useful source of cells for this process provided that collection and ex vivo expansion of hematopoietic stem and progenitor cells could be optimized. Here we investigated whether mesenchymal stem cells could sustain culture of irradiated peripheral blood CD34+ cells. In vitro irradiated (4 Gy 60Co gamma rays) or nonirradiated mobilized peripheral blood CD34+ cells from baboons were cultured for 7 days in a serum-free medium supplemented with stem cell factor+thrombopoietin+interleukin 3+FLT3 ligand (50 ng/ml each) in the presence or absence of mesenchymal stem cells. In contrast to cultures without mesenchymal stem cells, irradiated CD34+ cells cultured with mesenchymal stem cells displayed cell amplification, i.e. CD34+ (4.9-fold), CD34++ (3.8-fold), CD34++/Thy-1+ (8.1-fold), CD41+ (12.4-fold) and MPO+ (50.6-fold), although at lower levels than in nonirradiated CD34+ cells. Fourteen times more clonogenic cells, especially BFU-E, were preserved when irradiated cells were cultured on mesenchymal stem cells. Moreover, we showed that the effect of mesenchymal stem cells is related mainly to the reduction of apoptosis and involves cell-cell contact rather than production of soluble factor(s). This experimental model suggests that mesenchymal stem cells could provide a crucial tool for autologous cell therapy applied to accidentally irradiated victims.     

白细胞介素4降低脐带间充质干细胞对造血干细胞分化潜能的维持能力

目的探讨白细胞介素4（IL-4）对脐带间充质干细胞（UC-MSC）维持造血干细胞分化的影响。 方法将UC-MSC与脐带血CD34⁺造血干细胞按照造血支持能力常用的方案共培养,实验分为对照组和IL-4组,IL-4处理组加入IL-4（20 ng/ml）培养14 d。收集细胞并计数,使用流式细胞仪检测表达CD34的细胞比例。取3×10³个细胞,加入到半固体培养基,培养14 d后,通过倒置显微镜观察比较各种集落的形成,并使用流式细胞仪分析其中巨噬细胞和粒细胞表面特异性蛋白CD11b、CD14和CD15的表达。对两独立样本进行t检验统计学分析。 结果加入IL-4后,共培养体系中细胞数量（1.31±0.05）×10⁵个/孔与对照组（2.80±0.28）×10^5?个/孔相比下降,差异有统计学意义（t = 7.31,P < 0.05）,并且流式细胞分析显示其中的CD34⁺细胞比例也有降低。3×10³个IL-4组得到的细胞形成巨噬细胞集落形成单位的能力（9.33±1.53）?个/孔较对照组（17.67±0.58）个/孔有明显下降,差异有统计学意义（t = 8.84,P?< 0.001）;形成粒-巨噬集落形成单位的能力（15.67±3.22）个/孔较对照组（29.33±4.04）?个/?孔有明显下降,差异有统计学意义（t = 4.58,P < 0.05）;形成总集落单位的能力（39.33±9.07）个/?孔较对照组（62.67±6.66）?个/?孔也有下降,差异有统计学意义（t = 3.59,P < 0.05）。IL-4组得到的细胞分化出的细胞总数（3.67±1.71）×10⁵个/孔与对照组（9.50± 3.13）×10⁵个/孔相比也明显下降,差异有统计学意义（t = 2.83,P < 0.05）,而流式细胞术分析发现分化成的细胞中CD14⁺细胞比例也下降。 结论IL-4可以降低UC-MSC对造血干细胞分化潜能的维持能力,提示在Ⅱ型辅助T细胞相关体液免疫疾病中使用间充质干细胞治疗时,也需要兼顾机体造血相关功能。     

Importance of CD34+ cell subsets in autologous PBSC transplantation: the mulhouse experience using CD34+CD38- cells as predictive tool for hematopoietic engraftment

Over the years, various biological parameters have been proposed for predicting rapidity and long term maintenance of hematopoietic engraftment after peripheral blood stem cell transplantation (PBSCT). Determination of the graft content in CFU-GM was the only one available until the end of the eighties. But, for technical reasons, and also because it does not actually evaluate the self-renewal potential of the cell products reinfused, it has now been commonly replaced by the determination of CD34+ cell amounts, which are known to contain the pluripotent hematopoietic stem cells. However, a frequent discrepancy still exists between the number of CD34+ cells reinfused and the engraftment efficiency. We have recently demonstrated a higher accuracy of the numbers of CD34+38- cells contained in graft products to predict rapidity of trilineage engraftment, which has further been confirmed by other investigators. Furthermore, we and others, have proposed a threshold dose of 5 x 10(4) CD34+38- cells/kg b.w. below which the trilineage engraftment kinetics are significantly slower and unpredictible. This "cut-off" value also appears to be a realistic clinical tool to decide if hematopoietic growth factor(s) must be administered or not after PBSCT. Indeed, when for example, rh-G-CSF administration after transplant of CD34+38- amounts < 5 x 10(4) kg has indisputable positive effects on the rapidity of neutrophil engraftment, length of hospitalization and posttransplant costs, enough to make it fully justified in this situation, it is absolutely not the case when it is administered after reinfusion of CD34+38- cell amounts > 5 x 10(4) /kg. In this case, posttransplant rh-G-CSF administration could even result in a decrease in stem cells with self-renewal potential of the graft, which should still raise more concerns for its indiscriminate and costly use.     

Preadipocytes in the human subcutaneous adipose tissue display distinct features from the adult mesenchymal and hematopoietic stem cells

The stroma-vascular fraction (SVF) of human adipose tissue has recently been described to be composed of endothelial cells identified as CD34+/CD31+ cells, infiltrated/resident macrophages defined as CD14+/CD31+ cells, and a new cell population characterized as CD34+/CD31- cells. To elucidate the cell identity of the adipocyte precursor cells, fluorescent activating cell sorter (FACS) analyses were performed on crude SVF cultured under adipogenic conditions, i.e., serum-deprived medium containing insulin, cortisol, triiodothyronine, and supplemented with a PPARgamma agonist for the first 3 days. The progressive accumulation of lipid droplets was associated with a selective enrichment of the CD34+/CD31- cell population whereas control experiments performed in medium supplemented with 10% serum showed an overall downregulation of the three cell markers without adipogenesis. Among the different cell subsets, the CD34+/CD31- subset was the unique cell fraction able to answer to adipogenic culture conditions. Indeed, a time-dependent expression of adipocyte markers as well as acquisition of adipocyte-typical metabolic activities were observed. In parallel, the gene expression of lipogenic and lipolytic enzymes increased. The ability to differentiate into adipocytes was restricted to cells that did not express the mesenchymal stem cell marker CD105. Furthermore, the CD34+/CD31- cells did not respond to culture conditions used for hematopoietic colony assays. Taken together, the present study demonstrates that adipocyte progenitor cells, i.e., the preadipocytes, are included in the CD34+/CD31- cell fraction, which displays distinct features from the adult mesenchymal and hematopoietic stem cells.     

Pre-Transplantation Serum Parathyroid Hormone Influences the Number of Mobilized CD34+ Hematopoietic Stem Cells in Autologous Hematopoietic Stem Cell Transplantation

Background:Parathyroid hormone (PTH) is a calcium homeostasis regulator and can affect bone marrow niche. PTH leads to the bone marrow stem cell niche expansion as well as the induction of stem cell mobilization from the bone marrow into peripheral blood. In this study, we evaluated the association between pre- transplantation serum PTH levels and the number of circulating CD34+ cells along with the platelets/white blood cells (Plt/WBC) engraftment in patients who underwent autologous Hematopoietic Stem Cell Transplantation.Methods:Subjects for the study were 100 patients who received autologous hematopoietic stem cell transplantation (auto-HSCT), retrospectively. Serum levels of PTH, calcium, phosphorus, and alkaline phosphatase were measured before mobilization. Their impacts were measured on the number of mobilized CD34+ hematopoietic stem cells, and Plt/WBC engraftment.Results:High levels of serum PTH (> 63.10 pg/mL) was significantly associated with higher number of CD34+ cells in peripheral blood after granulocyte- colony stimulating factor (G-CSF)-induced mobilization (p= 0.079*). Serum calcium at low levels were associated with higher number of circulating CD34+ cells post mobilization. Pre- transplantation serum levels of phosphorus and alkaline phosphatase on CD34+ numbers were not statistically significant. Serum Plt/WBC engraftment was not improved in presence of high levels of serum PTH.Conclusion:We suggested that serum PTH levels before transplantation could be influential in raising the number of circulating CD34+ hematopoietic stem cell after mobilization.Key Words: Auto-HSCT, CD34+ Cell, Pre- transplant PTH     

Genetically modified CD34+ cells do not contribute to the mesenchymal compartment after autologous transplantation in the baboon

BACKGROUND: There is ongoing controversy about the transdifferentiation of hematopoietic stem cells (HSC) into different tissues such as mesenchymal cells. This transdifferentiation or 'plasticity' would be an appealing concept for many therapeutic strategies. While studies in the murine model show encouraging results, reports from clinical allogeneic stem cell transplantations do not support the concept of HSC plasticity. Our aim was to determine whether transplantation of transduced autologous marrow CD34+ cells leads to long-term engraftment of gene-marked cells with mesenchymal characteristics in the baboon. METHODS: We analyzed marrow of two baboons that had received green fluorescence protein (GFP)-marked CD34+ autologous marrow cells after myeloablative conditioning. Marrow was obtained 1 and 2.5 years after transplantation and adherent CD11a- (pan-leukocyte Ab) cells were cultured for 3 weeks. Cultures were then analyzed by flow cytometry and fluorescence microscopy for the presence of GFP+ cells. For further analysis fresh and cultured cells were also labeled with multiple Ab and functional analysis was performed. RESULTS: Both animals showed persistent and stable GFP marking by flow cytometry in peripheral blood leukocytes as well as in CD34+ marrow cells at 1 and 2.5 years after transplantation. There was no evidence of GFP+ mesenchymal cells by either flow cytometry or fluorescence microscopy, while functional and phenotypical analysis identified mesenchymal stem cells in these cultures. DISCUSSION: We conclude that genetically modified CD34+ cells do not contribute to the adherent marrow-derived mesenchymal cell population after autologous transplantation.     

Analysis and cryopreservation of hematopoietic stem and progenitor cells from umbilical cord blood

BACKGROUND: Umbilical cord blood (UCB) is an important source of hematopoietic stem and progenitor cells (HSC/HPC) for the reconstitution of the hematopoietic system after clinical transplantation. Cryopreservation of these cells is critical for UCB banking and transplantation as well as for research applications by providing readily available specimens. The objective of this study was to optimize cryopreservation conditions for CD34+ HSC/HPC from UCB. METHODS: Cryopreservation of CD34+ HSC/HPC from UCB after mononuclear cell (MNC) preparation was tested in a research-scale setup. Experimental variations were concentration of the cryoprotectant, the protein additive and cell concentration. In addition, protocols involving slow, serial addition and removal of DMSO were compared with standard protocols (fast addition and removal of DMSO) in order to avoid osmotic stress for the cryopreserved cells. Viability and recoveries of MNC, CD34+ cells and total colony-forming units (CFU) were calculated as read-outs. In addition, sterility testing of the collected UCB units before further processing was performed. RESULTS: The optimal conditions for cryopreservation of CD34+ HPC in MNC preparations were 10% DMSO and 2% human albumin at high cell concentrations (5 x 10(7) MNC/mL) with fast addition and removal of DMSO. After cryopreservation using a computer-controlled freezer, high viabilities (89%) and recoveries for CD34+ cells (89%) as well as for CFU (88%) were observed. Microbial contamination of the collected UCB samples was reduced to a rate of 6.4%. DISCUSSION: Optimized cryopreservation conditions were developed for UCB MNC in respect of the composition of the cryosolution. In addition, our results showed that fast addition of DMSO is essential for improved cryopreservation and post-thaw quality assessment results, whereas the speed of DMSO removal after thawing has little influence on the recoveries of CD34+ cells and CFU.     

Identification and analysis of in vitro cultured CD45-positive cells capable of multi-lineage differentiation

We report on a subset of cells that co-purify with CD45-positive/Lineage minus (CD45(pos)/Lin(minus)) hematopoietic cells that are capable of in vitro differentiation into multi-potential cells including cells with neuroectoderm properties. Although these cells are CD45 positive and have properties similar to CD45-negative mesenchymal progenitor cells (MPC) derived from bone marrow (BM), they are neither hematopoietic cells nor mesenchymal cells. These CD45(pos)/Lin(minus) cells can be expanded in vitro, express the stem cell genes Oct-4 and Nanog and can be induced to differentiate into endothelial cells, osteoblasts, muscle cells and neural cells at frequencies similar to those reported for bone marrow mesenchymal cells. Long-term culture of these cells followed by transplantation into NOD/SCID mice resulted in positive bone marrow stromal cell engraftment but not hematopoietic engraftment, suggesting that despite their CD45-positive status these cells do not have the same properties as hematopoietic stem cells. Clonal cell analysis determined that the culture period caused a broadening in the differentiation potential of the starting population.     

Ex vivo expansion of cord blood mononuclear cells on mesenchymal stem cells

BACKGROUND: Cord blood (CB) cells are being used increasingly as a source of hematopoietic cells to support high dose chemotherapy. However, CB units contain low numbers of cells, including CD34+ cells, and thus their use is associated with significant delays in engraftment of neutrophils and platelets. Exvivo expansion of CB has been proposed to increase the numbers of cells available. We and others have reported the requirement of CD34 selection for optimal expansion of CB products'; however, the selection of frozen CB products in clinical trials results in significant loss of CD34+ cells, with a median recovery of 50, but less than 40% recovery in more than one-third of products. In the present studies we evaluated the potential of mesenchymal stem cells (MSC) to support ex vivo expansion of unselected CB products. METHODS: Mononuclear cells (MNC) from CB products were isolated and cultured on preformed MSC layers in T150 flasks containing 50 mL Stemline II media plus hematopoietic growth factors. Various culture conditions were compared for optimal expansion of the CB MNC. RESULTS: Ex vivo expansion of CB MNC on MSC resulted in 10- to 20-fold expansion of total nucleated cells, seven- to 18-fold expansion of committed progenitor cells, two- to five-fold expansion of primitive progenitor cells and 16- to 37-fold expansion of CD34+ cells. DISCUSSION: These studies demonstrated significant expansion of CB products without CD34 cell selection using culture conditions that are clinically applicable. Our current focus is to initiate clinical trials to evaluate the in vivo potential of CB cells expanded with these conditions.     

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.     

Specific marking of hESCs-derived hematopoietic lineage by WAS-promoter driven lentiviral vectors

Genetic manipulation of human embryonic stem cells (hESCs) is instrumental for tracing lineage commitment and to studying human development. Here we used hematopoietic-specific Wiskott-Aldrich syndrome gene (WAS)-promoter driven lentiviral vectors (LVs) to achieve highly specific gene expression in hESCs-derived hematopoietic cells. We first demonstrated that endogenous WAS gene was not expressed in undifferentiated hESCs but was evident in hemogenic progenitors (CD45(-)CD31(+)CD34(+)) and hematopoietic cells (CD45(+)). Accordingly, WAS-promoter driven LVs were unable to express the eGFP transgene in undifferentiated hESCs. eGFP(+) cells only appeared after embryoid body (EB) hematopoietic differentiation. The phenotypic analysis of the eGFP(+) cells showed marking of different subpopulations at different days of differentiation. At days 10-15, AWE LVs tag hemogenic and hematopoietic progenitors cells (CD45(-)CD31(+)CD34(dim) and CD45(+)CD31(+)CD34(dim)) emerging from hESCs and at day 22 its expression became restricted to mature hematopoietic cells (CD45(+)CD33(+)). Surprisingly, at day 10 of differentiation, the AWE vector also marked CD45(-)CD31(low/-)CD34(-) cells, a population that disappeared at later stages of differentiation. We showed that the eGFP(+)CD45(-)CD31(+) population generate 5 times more CD45(+) cells than the eGFP(-)CD45(-)CD31(+) indicating that the AWE vector was identifying a subpopulation inside the CD45(-)CD31(+) cells with higher hemogenic capacity. We also showed generation of CD45(+) cells from the eGFP(+)CD45(-)CD31(low/-)CD34(-) population but not from the eGFP(-)CD45(-)CD31(low/-)CD34(-) cells. This is, to our knowledge, the first report of a gene transfer vector which specifically labels hemogenic progenitors and hematopoietic cells emerging from hESCs. We propose the use of WAS-promoter driven LVs as a novel tool to studying human hematopoietic development.     

MERP1: a mammalian ependymin-related protein gene differentially expressed in hematopoietic cells

We have utilized differential display polymerase chain reaction to investigate the gene expression of hematopoietic progenitor cells from adult bone marrow and umbilical cord blood. A differentially expressed gene was identified in CD34+ hematopoietic progenitor cells, with low expression in CD34- cells. We have obtained the full coding sequence of this gene which we designated human mammalian ependymin-related protein 1 (MERP1). Expression of MERP1 was found in a variety of normal human tissues, and is 4- and 10-fold higher in adult bone marrow and umbilical cord blood CD34+ cells, respectively, compared to CD34- cells. Additionally, MERP1 expression in a hematopoietic stem cell enriched population was down-regulated with proliferation and differentiation. Conceptual translation of the MERP1 open reading frame reveals significant homology to two families of glycoprotein calcium-dependant cell adhesion molecules: ependymins and protocadherins.     

Forced expression of HoxB4 enhances hematopoietic differentiation by human embryonic stem cells

HoxB4 has been shown to enhance hematopoietic engraftment by hematopoietic stem cells (HSC) from differentiating mouse embryonic stem cell (mESC) cultures. Here we examined the effect of ectopic expression of HoxB4 in differentiated human embryonic stem cells (hESCs). Stable HoxB4-expressing hESCs were established by lentiviral transduction, and the forced expression of HoxB4 did not affect stem cell features. HoxB4-expressing hESC-derived CD34+ cells generated higher numbers of erythroid and blast-like colonies than controls. The number of CD34+ cells increased but CD45+ and KDR+ cell numbers were not significantly affected. When the hESC derived CD34+ cells were transplanted into NOD/SCID beta 2m-/- mice, the ectopic expression of HoxB4 did not alter their repopulating capacity. Our findings show that overexpression of HoxB4 in differentiating hESCs increases hematopoietic colony formation and hematopoietic cell formation in vitro, but does not affect in vivo repopulation in adult mice hosts.     

Transduction of CD34+ hematopoietic progenitor cells with an antitat gene protects T-cell and macrophage progeny from AIDS virus infection.

Transduction of hematopoietic stem cells with genes that inhibit human immunodeficiency virus (HIV) replication has the potential to reconstitute immune function in individuals with AIDS. We evaluated the ability of an autoregulated gene, antitat, to inhibit replication of simian immunodeficiency virus (SIV) and HIV type 1 (HIV-1) in hematopoietic cells derived from transduced progenitor cells. The antitat gene expresses an antiviral RNA encoding polymeric Tat activation response elements in combination with an antisense tat moiety under the control of the HIV-1 long terminal repeat. CD34+ hematopoietic progenitor cells were transduced with a retroviral vector containing the antitat gene and then cultured under conditions that support in vitro differentiation of T cells or macrophage-like cells. Rhesus macaque CD4+ T cells and macrophage-like cells derived from CD34+ bone marrow cells transduced with the antitat gene were highly resistant to challenge with SIV, reflecting a 2- to 3-log reduction in peak SIV replication compared with controls. Similarly, human CD4+ T cells derived from CD34+ cord blood cells transduced with antitat were also resistant to infection with HIV-1. No evidence for toxicity of the antitat gene was observed in any of five different lineages derived from transduced hematopoietic cells. These results demonstrate that a candidate therapeutic gene introduced into hematopoietic progenitor cells can retain the ability to inhibit AIDS virus replication following T-cell differentiation and support the potential use of the antitat gene for stem cell gene therapy.