High efficiency gene transfer to human CD34+ cells

We have previously reported the development of improved MLV-based retroviral vectors whose prototype is entitled MT (Kim et al, J. Virol. 72:994-1044; Yu et al, Gene Therapy 7:797-804). The MT vector does not contain any viral coding sequences, and thus the possibility of homologous recombination between the vector and the packaging genome is virtually nil. Indeed, in a shotgun RCR detection assay, an MT-based vector did not produce any RCR. On the contrary, the MFG vector, containing parts of all three viral coding sequences (gag, pol, and env), generated a significant number of RCR. In addition to being safe, MT-based vectors produce levels of gene expression and viral titer comparable to or higher than other vectors currently available within the community. Based on this vector, we have constructed a number of retroviral vectors that can be used for the treatment of a variety of human diseases. Our major target diseases are those that can be treated with or the status of which can be significantly improved with bone marrow transplantation. To obtain the most significant therapeutic effects, it is necessary to achieve the highest possible gene delivery efficiency, drive the highest level of gene expression, and prevent expression of the inserted therapeutic gene from being negatively influenced by the genome environment. To these ends, we compared various LTRs for their effects on the level of gene expression, tested the effect of cis-acting elements that may influence chromatin structure or position effect of the inserted gene, and studied different transduction conditions for their gene delivery efficiency. Data recently obtained from these experiments will be presented.     

Thrombopoietin, flt3, and kit ligands together suppress apoptosis of human mobilized CD34+ cells and recruit primitive CD34+ Thy-1+ cells into rapid division.

Various combinations of cytokines have profoundly different effects on inhibition of apoptosis and stimulation of self-renewal division of hematopoietic stem cells (HSC) in short-term, ex vivo culture. Our goal was to quantitate expansion of cells with a primitive CD34+ Thy-1+ phenotype, as well as cell cycling, division history, differentiation, and apoptosis of CD34+ cells enriched from normal donor mobilized peripheral blood (MPB) cells. The balance of these parameters determines the net number of transplantable HSC produced in ex vivo cultures. Comparing several different combinations of cytokines added to 90-hour cultures of MPB CD34 cells, thrombopoietin (TPO), flt3 ligand (FL), and c-kit ligand (KL) gave the best result, with the lowest percentage of apoptotic cells and a mean 1.2-fold increase in the number of CD34+ Thy-1+ cells. A combination of interleukin 3 (IL-3), interleukin 6 (IL-6), and leukemia inhibitory factor (LIF) gave the worst outcome, including a decrease of CD34+ Thy-1+ cell number to a mean of 30% of the starting cell number. Cell division history was tracked using the dye 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE). Division of CD34+ Thy-1+ cells was faster and more synchronous in TPO, FL, and KL than in IL-3, IL-6, and LIF, which left a significant proportion of CD34+ cells undivided. Such detailed analyses of short-term, ex vivo cultures generated "replication scores," which allowed prediction of a sixfold improvement of the efficiency of gene transduction of primitive hematopoietic progenitors from MPB, using TPO, FL, and KL to replace IL-3, IL-6, and LIF. Analysis of retroviral transduction efficiency confirmed the increase of transgene expression from MPB primitive hematopoietic progenitors assayed after stromal culture was fivefold, validating the usefulness of multiparameter analysis of short-term cultures for survival and replication of CD34+ Thy-1+ cells.     

Lentiviral gene transfer into peripheral blood-derived CD34+ NOD/SCID-repopulating cells.

This study reports a lentiviral gene transfer protocol for efficient transduction of adult human peripheral blood (PB)-derived CD34+ NOD/SCID-repopulating cells (SRCs) using vesicular stomatitis virus-G protein (VSV-G)-pseudotyped lentiviruses encoding for enhanced green fluorescence protein (eGFP). Lentiviral stocks were concentrated by anion exchange chromatography, and transduction was performed under serum-free conditions at a multiplicity of infection (MOI) between 3 and 50. Similar transduction efficiencies were achieved in the presence and absence of cytokines. Transduction of PB-derived CD34+ cells at a MOI of 3 resulted in gene transfer efficiencies into SRCs of 9.2% and 12.0% in the absence and presence of cytokines, respectively. Using improved lentiviral vectors, transduction frequency varied between 42.0% (MOI 10) and 36.0% (MOI 50) with multilineage transgene expression within SRC-derived myeloid and lymphoid cells. The protocol described can be adapted for clinical application of lentiviral gene transfer into PB-derived CD34+ cells from adult patients.     

Thrombopoietin stimulates megakaryocytopoiesis, myelopoiesis, and expansion of CD34+ progenitor cells from single CD34+Thy-1+Lin- primitive progenitor cells

Thrombopoietin (TPO) or MpI ligand is known to stimulate megakaryocyte (MK) proliferation and differentiation. To identify the earliest human hematopoietic cells on which TPO acts, we cultured single CD34+Thy- 1+Lin- adult bone marrow cells in the presence of TPO alone, with TPO and interleukin-3 (IL-3), or with TPO and c-kit ligand (KL) in the presence of a murine stromal cell line (Sys1). Two distinct growth morphologies were observed: expansion of up to 200 blast cells with subsequent differentiation to large refractile CD41b+ MKs within 3 weeks or expansion to 200-10,000 blast cells, up to 25% of which expressed CD34. The latter blast cell expansions occurred over a 3- to 6-week period without obvious MK differentiation. Morphological staining, analysis of surface marker expression, and colony formation analysis revealed that these populations consisted predominantly of cells committed to the myelomonocytic lineage. The addition of IL-3 to TPO-containing cultures increased the extent of proliferation of single cells, whereas addition of KL increased the percentage of CD34+ cells among the expanding cell populations. Production of multiple colony- forming unit-MK from single CD34+Thy-1+Lin- cells in the presence of TPO was also demonstrated. In limiting dilution assays of CD34+Lin- cells, TPO was found to increase the size and frequency of cobblestone areas at 4 weeks in stromal cultures in the presence of leukemia inhibitory factor and IL-6. In stroma-free cultures, TPO activated a quiescent CD34+Lin-Rhodamine 123lo subset of primitive hematopoietic progenitor cells into cycle, without loss of CD34 expression. These data demonstrate that TPO acts directly on and supports division of cells more primitive than those committed to the MK lineage.     

Transplantation of allogeneic CD34+ blood cells

Pluripotent stem cells of hematopoiesis and lymphopoiesis are among the CD34+ cells in blood or bone marrow. After granulocyte-colony stimulating factor (G-CSF) treatment, 1% to 2% of the mononuclear cells in blood are CD34+ cells, which can be procured by leukapheresis. We investigated the potential of CD34+ blood cells for reconstituting hematopoiesis and lymphopoiesis after allogeneic transplantation. HLA- identical sibling donors of 10 patients with hematologic malignancies were treated with G-CSF (filgrastim), 5 microgram/kg subcutaneously twice daily for 5 to 7 days. CD34+ cells were selected from the apheresis concentrates by immunoadsorption, concomitantly the number of T cells was reduced 100- to 1,000-fold. After transplantation, five patients received cyclosporine A for graft-versus-host disease (GvHD) prophylaxis (group I); five patients additionally received methotrexate (group II). G-CSF and erythropoietin were given to all patients. Mean numbers of 7.45 x 10(6) CD34+ and 1.2 x 10(6) CD3+ cells per kilogram were transplanted. In group I, the median times of neutrophil recovery to 100, 500, and 1,000 per mm3 were 10, 10, and 11 days, respectively. Group II patients reached these neutrophil levels after 10, 14, and 15 days, respectively. Platelet transfusions were administered for a median of 18 days in group I and 30 days in group II, and red blood cells for 9 and 12 days, respectively. Between day 30 and 60, lymphocytes reached levels of 353 +/- 269 cells per mm3. The median grades of acute GvHD were III in group I and I in group II. Two patients in group I died from acute GvHD. Two leukemic relapses occurred in group II. Complete and stable donor hematopoiesis was shown in all patients with a median follow up of 370 (45 to 481) days. Allogeneic blood CD34+ cells can successfully reconstitute hematopoiesis and lymphopoiesis. Reduction of T cells by CD34+ blood cell enrichment and cyclosporine A alone might not be sufficient for prophylaxis of severe acute GvHD.     

Transplantation of a fetus with paternal Thy-1(+)CD34(+)cells for chronic granulomatous disease

A fetus diagnosed with X-linked chronic granulomatous disease was transplanted with Thy-1(+)CD34(+) cells of paternal origin. The transplant was performed at 14 weeks gestation by ultrasound guided injection into the peritoneal cavity. The fetus was delivered at 38 weeks gestation after an otherwise uneventful pregnancy. Umbilical cord blood was collected and used to determine the level of peripheral blood chimerism as well as levels of functional engrafted cells. Flow cytometry was used to detect donor leukocytes identified as HLA-A2(-)B7(+) cells, whereas recipient cells were identified as HLA-A2(+)B7(-) cells. No evidence of donor cell engraftment above a level of 0.01% was found. PCR was used to detect HLA-DRB1*15(+) donor cells among the recipient's HLA-DRB1*15(-) cells, but no engraftment was seen with a sensitivity of 1:1000. The presence of functional, donor-derived neutrophils was assessed by flow cytometry using two different fluorescent dyes that measure reactive oxygen species generated by the phagocyte NADPH oxidase. No evidence of paternal-derived functional neutrophils above a level of 0.15% was observed. Peripheral blood and bone marrow samples were collected at 6 months of age. Neither sample showed engraftment by HLA typing using both flow cytometry and PCR. Functional phagocytes were also not observed. Furthermore, no indication of immunological tolerance specific for the donor cells was indicated by a mixed lymphocyte reaction assay performed at 6 months of age. While there appears to be no engraftment of the donor stem cells, the transplant caused no harm to the fetus and the child was healthy at 6 months of age. Analyses of fetal tissues, obtained from elective abortions, revealed that CD3(+) T cells and CD56(+)CD3(-) NK cells are present in the liver at 8 weeks gestation and in the blood by 9 weeks gestation. The presence of these lymphocytes may contribute to the lack of donor cell engraftment in the human fetus.     

Highly efficient gene transfer into mobilized CD34+ hematopoietic cells using serotype-5 adenoviral vectors and BoosterExpress Reagent

OBJECTIVE: To optimize transduction efficiency of mobilized CD34(+) cells with serotype-5 adenoviruses (Ad5s), we investigated the activity of the chemical cocktail BoosterExpress Reagent in enhancing Ad5-mediated gene transfer into CD34(+) cells. METHODS: Enriched CD34(+) cells were transduced with three different Ad5s at increasing multiplicity of infections (MOIs) in the presence and absence of BoosterExpress Reagent. Percentages of transduced cells and levels of transgene expression were quantified by flow cytometry. Propidium iodide staining and colony growth were used to assess the toxicity of the transduction protocol. Expression of adenovirus receptors was investigated by flow cytometry. RESULTS: Irrespective of the Ad5 used, transduction with BoosterExpress Reagent using an MOI of 500 resulted in an average six- to seven-fold increase of transduction efficiencies and 1.5- to 2-fold increase of the levels of transgene expression, which could be detected up to 7 days post-transduction. Although BoosterExpress Reagent alone did not affect the plating efficiency of CD34(+) cells, adenovector transduction plus BoosterExpress Reagent significantly reduced the plating efficiency due to the marked increase of transduced cells. However, adenoviral transduction in the presence of BoosterExpress Reagent failed to significantly reduce the recovery of CD34(+) cells as compared with transduction in the absence of the compound. Coxsackievirus and adenovirus receptor as well as alpha(v)beta(3), alpha(v)beta(5), alpha(5), and beta(1) integrins were upregulated by BoosterExpress Reagent. CONCLUSIONS: BoosterExpress Reagent allows high-levels of durable transgene expression, thus making CD34(+) cells a suitable target for Ad5-mediated gene transfer.     

Transfer and expression of the human multiple drug resistance gene in human CD34+ cells

The human multiple-drug resistance (MDR1) gene has been transferred into human hematopoietic progenitors using retroviral gene transfer. Human bone marrow cells and isolated CD34+ cells isolated from marrow were exposed to growth factors interleukin-3 (IL-3), IL-6, and stem cell factor for 48 hours and then to two changes of MDR retroviral supernatants over the next 24 hours. Progenitor assays in methylcellulose at this time showed that 18% to 70% of BFU-E and 30% to 60% of CFU-GM contain the transferred MDR gene by polymerase chain reaction analysis. Up to 11.2% of the progeny of these cells express increased amounts of MDR glycoprotein on their surface by fluorescence- activated cell sorter (FACS) analysis. In addition, transduced cells are enriched in high MDR-expressing cells after exposure to taxol as assessed by FACS analysis, and by resistance of BFU-E to taxol (Bristol- Myers Squibb, Princeton, NJ). These studies indicate the feasibility of using MDR gene transfer as a means of enriching marrow for MDR- transduced cells. They also provide the basis of a phase 1 clinical protocol in patients with advanced cancers not involving the bone marrow for the use of MDR gene transfer as a means of protecting marrow cells, which normally express low levels of MDR, from the myelosuppressive effects of drugs like taxol.     

Enrichment of human hematopoietic stem cell activity in the CD34+Thy- 1+Lin- subpopulation from mobilized peripheral blood

The number of CD34+ cells in the peripheral blood of cancer patients is known to be increased following the administration of high dose chemotherapy and hematopoietic growth factors. These so-called peripheral blood stem cell grafts are now frequently used for autologous transplantation of patients with malignancies. In this report, we address the question of whether true long-term repopulating pluripotent hematopoietic stem cells (PHSC) are mobilized into peripheral blood following chemotherapy plus granulocyte/macrophage colony-stimulating factor (GM-CSF) or granulocyte colony-stimulating factor (G-CSF) mobilization. We have examined the presence of stem cells in mobilized peripheral blood (MPB) by using an antibody to the human Thy-1 molecule to stain the CD34+Lineage- (Lin-) population. The kinetics of mobilization of CD34+Thy-1+ Lin- cells into peripheral blood were studied, and the percentage of cells with this phenotype was found to vary widely depending on the day of leukapheresis. A CD34+Thy- 1+Lin- cell population, potentially containing PHSCs, was isolated by fluorescence activated cell sorting (FACS) and analyzed for activity. The multilineage differentiative capacity of this candidate stem cell- containing population in MPB was determined using an in vitro long-term culture system, in which cobblestone area formation was used as a means of detecting PHSCs. We also measured repopulating capacity by using two in vivo models in which severe combined immunodeficiency (SCID)-hu mice were implanted with human fetal bone or thymus grafts. Using these assays, we show that the highest frequency of cobblestone area-forming cells (CAFC) after 7 weeks of culture was observed in a subpopulation of CD34+Lin- cells, which expressed low levels of Thy-1. This cell population was capable of producing both B and myeloid cells, and maintaining CD34+Lin- cells in these long term cultures. Moreover, the CD34+Thy-1+Lin- cell subset possessed a higher ability to engraft and to demonstrate multilineage differentiative potential at 8 weeks in the SCID-hu bone assay. However, in the SCID-hu thymus model, both Thy-1+ and Thy-1- subpopulations were capable of donor T-cell engraftment at 6 weeks, suggesting the presence of cells capable of initiating T lymphopoiesis in both populations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Production of human glucocerebrosidase in mice after retroviral gene transfer into multipotential hematopoietic progenitor cells.

The human glucocerebrosidase (GC) gene has been transferred efficiently into spleen colony-forming unit (CFU-S) multipotential hematopoietic progenitor cells, and production of human GC RNA and protein has been achieved in transduced CFU-S colonies. High-titer retroviral vectors containing the human GC cDNA were constructed. Mouse bone marrow cells were stimulated with hematopoietic growth factors, infected by coculture with producer cells, and injected into lethally irradiated animals. Four vectors were compared with respect to gene-transfer efficiency into CFU-S progenitors. One vector (G vector) required high concentrations of interleukins 3 and 6 during stimulation and coculture for efficient transduction of CFU-S progenitors. The remaining three vectors (NTG, GTN, and GI vectors) transduced these progenitors at infection frequencies approaching 100% using low concentrations of hematopoietic growth factors to stimulate cell division prior to and during the infection. Vectors using the viral long terminal repeat enhancer/promoter to drive the human GC cDNA produced high levels of human GC RNA in the progeny of CFU-S progenitors after gene transfer. When an internal herpes simplex thymidine kinase promoter assisted by a mutant polyoma enhancer was used to drive the human GC cDNA (NTG vector), little or no human GC RNA was detected in transduced CFU-S colonies. All three vectors producing human GC RNA in CFU-S colonies can generate human GC as detected by immunochemical analysis of CFU-S colonies. NTG vector-infected bone marrow cells were transplanted into W/Wv recipients to generate long-term reconstituted mice. The capacity of the viral long terminal repeat and the internal thymidine kinase promoter to direct synthesis of RNA in transduced bone marrow and spleen cells 5 months after bone marrow transplantation reflected the performance of these promoters in NTG-transduced CFU-S colonies.     

The AFT024 stromal cell line improves MDR1 gene transfer to CD34+ cells derived from human umbilical cord blood

Human hematopoietic stem cells (HSCs) are difficult to transfect with retroviral vectors because of their quiescent nature. Based on the theory that the murine fetal stromal cell line AFT024 can recruit significant numbers of HSC into cell cycle without loss of their primitive function, we transduced human umbilical cord blood cells (UCB) derived CD34+ cells with a retroviral vector pHaMDR1/A containing the human multidrug resistant 1 gene (MDR1) during co-culture with the AFT024 feeders. We found that the presence of the AFT024 cells increased the proportion of Rh-123dull cells up to 35.5%+/-11.4% and transduced colony-forming cells (CFCs) up to 15.2%. Six weeks after transplantation of 5x10(4) day 0 uncultured CD34+ HSCs or their equivalents expanded in the presence or absence of the AFT024 cells for 21 days into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice, we found that CD34+ cells expanded in the presence of the AFT024 cells engrafted in each receptor mouse and the percentage of CD45+ cells reached 18.8%+/-9.5%, of which 18.1%+/-6.0% were Rh-123dull cells. These results suggest that the AFT024 stromal cells can significantly improve MDR1 gene transfer efficiency and maintain the engrafting ability of the CD34+ HSCs derived from UCB.     

Large-scale feasibility of gene transduction into human CD34+ cell-derived dendritic cells by adenoviral/polycation complex

With a view to using multiple injections of anti-cancer dendritic cell (DC)-based vaccines, we evaluated the feasibility of the adenoviral transduction of large amounts of human CD34+ cell-derived DCs, and analysed the persistence of the transgene expression and the integrity of DC functional activity after the transduction/cryopreservation procedures. Mature DCs generated from highly enriched human CD34+ cells were transduced by a recombinant adenovirus (rAd-MFG) that carried a modified, membrane-exposed, alkaline phosphatase (AP) sequence as the reporter gene. Cationic lipids such as LipofectAmine or poly-L-lysine were mixed with the viral particles before the transduction of the target cells. The highest transduction efficiency was obtained at a multiplicity of infection (MOI) rate of 500 (AP + DCs: 50 +/- 2%, viability =95%) under both small- and large-scale conditions. The addition of poly-L-lysine or LipofectAmine increased the percentage of transduced cells at an MOI of 500 (CD1a+/AP+ cells = 85 +/- 3% and 80 +/- 2% respectively). Polycations made it possible to reduce the amounts of viral particles, with high efficiency of transduction being achieved at a MOI of 100 with 10 microg/ml poly-L-lysine (CD1a+/AP+: 68 +/- 9%) or 30 microg/ml LipofectAmine (CD1a+/AP+: 60 +/- 7%). Evaluation of the immunophenotype of the transduced DCs showed that the lack of a DC subpopulation was more susceptible to adenoviral transduction. Cryopreservation of transduced DCs did not modify the viability or percentage of AP+ cells that maintain antigen-presenting cell (APC) functions. These findings indicate the efficacy of this method for the transduction of large amounts of CD34+ cell-derived DCs using small quantities of adenoviral vector mixed with polycations. Cryopreservation of transduced DCs did not damage their viability or APC functions, thus making it possible to plan multiple injections of engineered DC-based vaccines.     

Transplantation of a combination of CD133+ and CD34+ selected progenitor cells from alternative donors

Positive selected haematopoietic stem cells are increasingly used for allogeneic transplantation with the CD34 antigen employed in most separation techniques. However, the recently described pentaspan molecule CD133 appears to be a marker of more primitive haematopoietic progenitors. Here we report our experience with a new CD133-based selection method in 10 paediatric patients with matched unrelated (n = 2) or mismatched-related donors (n = 8). These patients received a combination of stem cells (median = 29.3 x 10(6)/kg), selected with either anti-CD34 or anti-CD133 coated microbeads. The proportion of CD133+ selected cells was gradually increased from patient to patient from 10% to 100%. Comparison of CD133+ and CD34+ separation procedures revealed similar purity and recovery of target populations but a lower depletion of T cells by CD133+ selection (3.7 log vs. 4.1 log, P < 0.001). Both separation procedures produced >90% CD34+/CD133+ double positive target cells. Engraftment occurred in all patients (sustained primary, n = 8; after reconditioning, n = 2). No primary acute graft versus host disease (GvHD) >/= grade II or chronic GvHD was observed. The patients showed a rapid platelet recovery (median time to independence from substitution = 13.5 d), whereas T cell regeneration was variable. Five patients are alive with a median follow-up of 10 months. Our data demonstrates the feasibility of CD133+ selection for transplantation from alternative donors and encourages further trials with total CD133+ separated grafts.     

Stable gene transfer to human CD34(+) hematopoietic cells using the Sleeping Beauty transposon

OBJECTIVE: Methods of gene transfer to hematopoietic stem cells that result in stable integration may provide treatments for many inherited and acquired blood diseases. It has been demonstrated previously that a gene delivery system based on the Sleeping Beauty (SB) transposon can be derived where a plasmid transiently expressing the SB transposase can mediate the stable chromosomal integration of a codelivered second plasmid containing a gene expression unit flanked by the inverted repeats derived from the transposon. METHODS: Plasmid DNA containing the elements required for SB transposition was delivered to hematopoietic cells via electroporation. Integrated transgene (enhanced green fluorescent protein [eGFP]) expression was assessed in vitro and in vivo. RESULTS: In the K562 human hematopoietic cell line, we observed stable expression of eGFP in >60% of cells for over 2 months after electroporation of the two plasmids; in contrast, in control cells either not treated with transposase or exposed to a defective mutant transposase, the level of gene expression had fallen to near background (<0.1%) by 2 weeks. In purified human cord blood CD34(+) progenitor cells, the transposase led to stable gene transfer at levels up to 6% for over 4 weeks, but gene transfer to more primitive nonobese diabetic/severe combined immunodeficient repopulating cells or CD34(+)/CD38(-) in long-term culture was low and electroporation of the cells with plasmid DNA caused significant cell death. CONCLUSION: The long-term stable expression highlights the potential of this transposase-based gene delivery method for ameliorating diseases affecting the hematopoietic system, although further improvements in gene transfer efficacy are needed.     

Expression and function of adhesion molecules on human hematopoietic stem cells: CD34+ LFA-1- cells are more primitive than CD34+ LFA-1+ cells.

Advances in fluorescence-activated cell sorter technology have brought about multicolor analysis of cell phenotypes. To clarify the phenotypes of human hematopoietic stem cells (HSCs), we initially prepared novel antibodies against CD34 and labeled one of them (4A1) with allophycocyanin (APC). With this, we analyzed the phenotypes of CD34+ HSCs and showed that primitive HSCs or CD34+CD33- cells expressed adhesion molecules such as CD43, CD44, CD11a, CD11c, CD18, and leukocyte adhesion molecule (LAM-1). The more primitive hematopoietic cells or CD34+CD38- cells also expressed CD11a and CD18 with an incidence of 20% to 30%. To clarify the role of adhesion molecules in HSCs, we examined the colony forming capacity after long-term culture with allogeneic irradiated stromal layers. Among CD34+CD33- cells, CD18+ cells gave rise to colony-forming cells (CFCs) on stromal layers, but reached a maximum at week 2, after which the number of generated CFCs decreased. On the other hand, CD18- cells generated less CFCs than CD18+ cells at 2 to 3 weeks, but increased after 4 weeks of culture. When CD18 or CD11a antibody was added to a coculture system of CD34+CD33- cells with stromal layers, the number of generated CFCs decreased significantly compared with the no antibody control. Leukocyte function-associated antigen-1 (LFA-1) (CD11a/CD18) was expressed on some populations of hematopoietic cells and contributed to the proliferation by interacting with stromal cells. However, more primitive cells capable of reconstituting hematopoiesis did not express LFA-1. These data provide a rationale for the administration of anti-LFA-1 antibody after bone marrow transplantation for reducing the graft failure.     

Characterization of Thy-1 (CDw90) expression in CD34+ acute leukemia

Thy-1 (CDw90) is a phosphatidylinositol-anchored cell surface molecule which, when coexpressed with CD34 in normal human bone marrow, identifies a population of immature cells that includes putative hematopoietic stem cells. To date, the characterization of Thy-1 expression has been confined largely to normal tissues and cell lines. In this study, we evaluated the frequency and intensity of Thy-1 expression as defined by reactivity with the anti-Thy-1 antibody 5E10 in 38 cases of CD34+ acute leukemia (21 acute myelogenous leukemia [AML], 8 chronic myelogenous leukemia [CML] in blast crisis, and 9 acute lymphoblastic leukemia [ALL]). In 34 of 38 cases (89%) the CD34+ cells lacked expression of the Thy-1 antigen. High-density Thy-1 expression was found in 1 case of CML in lymphoid blast crisis, and low- density Thy-1 expression was identified on a portion of the leukemic cells in 2 cases of AML with myelodysplastic features, and 1 case of CML in myeloid blast crisis, suggesting a possible correlation between Thy-1 expression and certain instances of stem cell disorders such as CML and AML with dysplastic features. In contrast, the dissociation of Thy-1 and CD34 expression in the majority of acute leukemias studied suggests that the development of these leukemias occurs at a later stage than the hematopoietic stem cell. Characterization of Thy-1 expression in acute leukemia may eventually provide insights into the origin of the disease. In addition, separation of leukemic blasts from normal stem cells based on Thy-1 expression may prove useful in assessing residual disease, as well as in excluding leukemic blasts from stem cell preparations destined for autologous bone marrow or peripheral stem cell transplantation.     

Aorta-associated CD34+ hematopoietic cells in the early human embryo

Hematopoiesis is established from circulating blood stem cells that seed the embryonic rudiments of blood-forming tissues, a basic notion in developmental hematology. However, the assumption that these stem cells originate from the extraembryonic mesoderm, where primitive hematopoiesis is initiated by intrinsic precursors, has been reconsidered after analysis of blood cell development in avian embryo chimeras: yolk-sac-derived stem cells do not contribute significantly to the definitive blood system, whose first forerunners develop independently along the ventral aspect of the embryonic aorta. Recently, the homologous intraembryonic tissues of the mouse have been submitted to sensitive in vivo and in vitro assays, which showed that they also harbor multipotential hematopoietic stem cells. We have now identified a dense population of hematogenous cells, marked by the surface expression of the CD34 glycoprotein, associated with the ventral endothelium of the aorta in the 5-week human embryo. Therefore, we extend to the human species the growing evidence that intraembryonic hematopoietic cells developing independently of the yolk sac might be the real stem of the whole blood system.