Efficient retrovirus-mediated transfer of the multidrug resistance 1 gene into autologous human long-term repopulating hematopoietic stem cells.

Pre-clinical studies indicate that efficient retrovirus-mediated gene transfer into hematopoietic stem cells and progenitor cells can be achieved by co-localizing retroviral particles and target cells on specific adhesion domains of fibronectin. In this pilot study, we used this technique to transfer the human multidrug resistance 1 gene into stem and progenitor cells of patients with germ cell tumors undergoing autologous transplantation. There was efficient gene transfer into stem and progenitor cells in the presence of recombinant fibronectin fragment CH-296. The infusion of these cells was associated with no harmful effects and led to prompt hematopoietic recovery. There was in vivo vector expression, but it may have been limited by the high rate of aberrant splicing of the multidrug resistance 1 gene in the vector. Gene marking has persisted more than a year at levels higher than previously reported in humans.

Antitumor activity and immunotherapeutic properties of Flt3-ligand in a murine breast cancer model.

Flt3-Ligand (Flt3-L) is a stimulatory cytokine for a variety of hematopoietic lineages, including dendritic cells and B cells. The antitumor properties of Flt3-L were evaluated in C3H/HeN mice challenged with the syngeneic C3L5 murine breast cancer cell line. Eighty % of animals receiving 500 microg/kg/day of Chinese hamster ovary-derived human Flt3-L for 10 days were protected from tumor growth, whether the tumor challenge was administered on the first or fourth days of Flt3-L administration. The protection provided by soluble Flt3-L was transient. All tumor-free animals rechallenged 4 weeks after the primary challenge developed tumor. Transduction of C3L5 with retroviral vectors expressing human or murine Flt3-L did not influence in vitro growth or MHC expression but decreased in vivo tumor development to 0 and 10% of mice, respectively. This compares with tumor growth of 52% with interleukin-2 transduced C3L5 and over 85% with untransduced and control vector-transduced C3L5. Unlike animals treated with soluble Flt3-L, administration of Flt3-L as a tumor vaccine protected mice from a subsequent challenge with untransduced C3L5 in 60-78% of mice, compared to 0% of controls. Our initial work used the most common Flt3-L isoform, which is membrane bound but can undergo proteolytic cleavage to generate a soluble form. To evaluate the role of the various Flt3-L isoforms in preventing tumor formation, retroviral vectors encoding only the membrane-bound form or only the soluble isoform were evaluated in the C3L5 model. Tumor formation was similar with either isoform, preventing tumor formation in 80-90% of mice after the primary challenge and 88-89% after the secondary challenge. Splenocytes obtained 4 weeks after the secondary challenge conferred adoptive immunity to naive mice in 60% of animals. This initial report of antitumor activity by Flt3-L is consistent with its known stimulatory effect on antigen-presenting cells and suggests it may enhance the development of tumor vaccines.

The 30/35 kDa chymotryptic fragment of fibronectin enhances retroviral-mediated gene transfer in purified chronic myelogenous leukemia bone marrow progenitors.

We have previously shown by reverse transcriptase-PCR (rtPCR) that CML CD34+ HLA-DR- cells are enriched for BCR/ABL(-) hematopoietic progenitor cells (HPC) while leukemic HPC reside predominately within CML CD34+ HLA-DR+ cells. We investigated whether the 30/35 kDa fragment of fibronectin (FN) could be used to enhance retroviral-mediated gene transfer (RMGT) in chronic phase CML marrow HPC. CML CD34+ HLA-DR- and CD34+ HLA-DR+ cells were transduced with vector supernate containing the neomycin resistance gene on plates coated with either FN or bovine serum albumin (BSA) as control, then assayed for transduced HPC in progenitor cell assays in the presence or absence of G418. Transduction efficiency of CML CD34+ HLA-DR- cells over BSA ranged from 0.09 to 7.2% (mean 3.3 +/- 1.5%), while that over FN plates ranged from 3.8 to 23% (mean 11.0 +/- 4.5%) (n = 4). Transduction efficiencies of CML CD34+ HLA-DR+ cells ranged from 0.4 to 9.8% (mean 3.7 +/- 1.7%) and 6.0 to 26% (mean 17.3 +/- 4.5%) (n = 5) over BSA and FN, respectively. rtPCR analysis for BCR/ABL mRNA of individual G418-resistant HPC generated from CD34+ HLA-DR- cells revealed that normal BCR/ABL(-) HPC were successfully transduced under these experimental conditions. These results demonstrate the feasibility of transducing normal CML primitive HPC, and illustrate the potential clinical use of FN in the setting of gene therapy for CML, as well as other diseases.

Homing, cell cycle kinetics and fate of transplanted hematopoietic stem cells.

Homing of transplanted hematopoietic stem cells to recipient bone marrow is a critical step in engraftment and initiation of marrow reconstitution. At present, only partial understanding of the cellular and molecular mechanisms governing homing exists. Likewise, only an incomplete list of adhesion molecules implicated in directing the trafficking of stem cells to the marrow microenvironment is available. Opposing hypotheses that attribute homing to an orderly and orchestrated cascade of events or to random migration of circulating cells find ample experimental support. Also unsettled is the fate of marrow-homed cells shortly after transplantation and the rapidity at which they begin to proliferate in their new marrow microenvironment. The limited number of studies in this field and disparities in their experimental design intensifies the confusion surrounding these critical aspects of stem cell biology. However, this area of research is moving forward rapidly and results capable of clarifying many of these issues are forthcoming.

Rapid exit from G0/G1 phases of cell cycle in response to stem cell factor confers on umbilical cord blood CD34+ cells an enhanced ex vivo expansion potential.

Currently, the most commonly used grafts of progenitor and stem cells for patients undergoing bone marrow transplantation (BMT) are derived from large collections of autologous or allogeneic adult human bone marrow (BM). The feasibility of using human umbilical cord blood (HUCB), normal peripheral blood (PB), and smaller collections of BM as sources of hematopoietic stem cell grafts for adult patients remains questionable. We investigated the ex vivo proliferative potential of HUCB CD34+ cells as a means of expanding HUCB grafts, thereby making them more acceptable for clinical transplantation. HUCB-derived CD34+HLA-DR+ cells, maintained for 5 days in suspension cultures supplemented with 10% HUCB plasma and a combination of stem cell factor (SCF) and interleukin-3 (IL-3), displayed a 10-fold increase in the total number of CD34+ cells. In contrast, only a four-fold increase was observed in identical cultures initiated with BM-derived CD34+HLA-DR+ cells. Whereas BM CD34+ cells failed to proliferate in response to SCF alone, HUCB CD34+ cells expanded 5.6-fold by day 5, thus demonstrating an enhanced response to SCF. When the effects of SCF on the exit of HUCB cells from G0/G1 phases of cell cycle were investigated, we found that although HUCB CD34+HLA-DR+ cells were more quiescent than BM CD34+HLA-DR+ and BM CD34+HLA-DR- cells (97.5% of HUCB CD34+HLA-DR+ in G0/G1 vs. 88.6% of BM CD34+HLA-DR+ and 92.0% of BM CD34+HLA-DR- [p < 0.005]), HUCB CD34+HLA-DR+ cells exited from dormancy more rapidly than BM cells, such that by 36 to 48 hours following exposure to SCF, only 55% remained in G0/G1. Furthermore, an 8.4-fold increase in the number of HUCB CD34+ cells still residing in G0/G1 was observed on day 5 in cultures supplemented with SCF and IL-3, suggesting the generation of large numbers of primitive hematopoietic progenitor cells (HPC) in vitro. When the contribution of HUCB plasma to the exist of HUCB CD34+HLA-DR+ cells from G0/G1 phases of cell cycle was investigated, it was found that in serum-free media supplemented with only SCF or IL-3, HUCB cells did not exist G0/G1 as rapidly as when HUCB plasma or SCF plus IL-3 was present. In contrast, when HUCB plasma was added to any cytokine combination, it did not enhance the exist of BM CD34+HLA-DR+ cells from G0/G1 phases of cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

Proliferation-induced decline of primitive hematopoietic progenitor cell activity is coupled with an increase in apoptosis of ex vivo expanded CD34+ cells.

We examined the decline in hematopoietic potential observed when human CD34+ cells are cultured in vitro by evaluating the association between proliferation history and the fate of long-term hematopoietic culture-initiating cells (LTHC-ICs) as well as the onset of programmed cell death. The membrane dye PKH2 was used to track ex vivo expanded human CD34+ cells from bone marrow, cord blood, and mobilized peripheral blood, and to identify and isolate CD34+ cells that had divided once, twice, three, or four times or more, as well as cells that had remained cytokine nonresponsive and therefore failed to proliferate. These isolated groups of cells were assayed for their hematopoietic potential, cell cycle status, and percentage of apoptotic cells. A gradual decline in the content of LTHC-ICs, as well as in their ability to initiate and sustain in vitro hematopoiesis, was found to correlate with the number of in vitro cellular divisions, such that the hematopoietic potential of CD34+ cells dividing four or more times was nearly depleted. DNA analysis revealed that cells dividing more than three times resided predominantly in G0/G1 phases of the cell cycle. In addition, the percentage of CD34+ cells undergoing apoptosis was found to increase concomitantly with the number of in vitro cellular divisions; less than 10% of cells dividing once were apoptotic, whereas more than 25% of CD34+ cells dividing four or more times underwent programmed cell death. Together, these data suggest that a proliferation-associated, and possibly activation-induced, loss of hematopoietic potential among dividing CD34+ cells may result from an increase in programmed cell death among dividing primitive hematopoietic progenitor cells.

Ex vivo expansion of murine hematopoietic progenitor cells generates classes of expanded cells possessing different levels of bone marrow repopulating potential.

The objective of ex vivo expansion of primitive hematopoietic progenitor cells (HPC) is to increase the number of progeny cells possessing hematopoietic potential similar to the original HPC. In the context of bone marrow (BM) transplantation in mice, this implies that expanding a number of HPC sufficient for long-term rescue of one lethally irradiated animal should generate enough cells to rescue more than one lethally irradiated recipient. In the present study, Sca-1+Lin- cells from male C57Bl/6 mice were expanded in vitro with stem cell factor (SCF), interleukin-1alpha (IL-1alpha), IL-3, and IL-6 and used to transplant lethally irradiated syngeneic female recipients. Expanded cells were tracked in vitro with the fluorescent membrane dye PKH2, which becomes evenly distributed among dividing daughter cells, and fractionated on day 7 into Sca-1+ cells which did not divide (Sca-1+PKH2bright), those which had divided 1 to 2 times (Sca-1+PKH2moderate), or those which had divided four or more times (Sca-1+PKH2dim). Grafts of expanded cells consisted of either the same number of fresh cells proven to rescue lethally irradiated animals [3X10(3) cells; referred to as one repopulating dose (1 RD)] or the expansion equivalent (EE) of these cells. One EE of cells represented 3X10(3) multiplied by the fold increase in the number of cultured cells on day 7. All animals transplanted with 3X10(3) freshly isolated Sca-1+Lin- cells survived long-term. Only 53% of animals receiving 1 EE of all cultured day-7 cells survived. One RD from all three PKH2 fractions (bright, moderate, and dim) of day-7 cultured Sca-1+ cells failed to rescue more than 30% of lethally irradiated recipients. Comparable survival rates were obtained when 1 EE of Sca-1+PKH2dim or only 4 RD of Sca-1+PKH2bright cells were used as grafts, suggesting that a larger frequency of long-term repopulating cells may have been retained within the fraction of Sca-1+ cells undergoing minimal or no proliferation in culture. Engraftment of male ex vivo expanded cells in recipients was confirmed by polymerase chain reaction (PCR) analysis with Y chromosome-specific primers. When analyzed for their cell cycle status, Sca-1+PKH2bright cells were mostly quiescent, whereas a higher percentage of Sca-1+PKH2dim cells were in active phases of cell cycle. These data suggest that ex vivo expansion does not augment the number of BM repopulating HPC and that ex vivo expansion generates classes of progenitor cells with different BM repopulating potentials depending on their proliferative history. These studies also suggest that the cell cycle status of graft cells may affect the ability of these cells to engraft in myeloablated hosts.

Effects of CD34+ selection and T cell immunodepletion on cord blood hematopoietic progenitors: relevance to stem cell transplantation.

Cord blood (CB) has been used recently for stem cell transplantation. We have investigated two different approaches to deplete CB samples of T cells capable of mounting graft-vs.-host disease (GVHD). The methods used were selection of CD34+ cells using avidin-biotin columns (CellPro) and T cell immunodepletion with T10B9 monoclonal antibody (mAb) plus complement. Using the avidin-biotin columns, 10.3% of the original CD34+ cells were recovered. Although this technique yielded a population containing 60 +/- 5.5% CD34+ cells, about 1 log of CFU-GM progenitors were lost. In contrast, after the T10B9 mAb and complement immunodepletion, 75 +/- 19% and 62 +/- 7% of the CD34+ cells and CFU-GM were recovered, respectively. T cell depletion was 3.6 logs using the CellPro columns and 2.2 logs after immunodepletion. To investigate whether cell losses following T cell depletion could be overcome by ex vivo expansion, cells were cultured in the presence of recombinant human interleukin-3 (rhIL-3) and recombinant human c-kit ligand (stem cell factor [rhSCF]) for 7 days. There were 14- and six-fold expansions in the number of progenitors recovered after CellPro and immunodepletion, respectively. To asses the engraftment potential of expanded cells, we used a murine transplantation model in which the presence of human cells was identified by the anti-CD45 mAb. Cells expanded in vitro engrafted in irradiated BNXid mice as efficiently as nonexpanded cells, suggesting that expansion did not affect their transplantability. This study shows that both techniques resulted in significant T cell depletion of CB. Furthermore, in vitro expansion could overcome cell losses sustained during the separation techniques without impairing the engraftment potential of the expanded cells.

Evaluation of the in vitro behavior of phenotypically defined populations of umbilical cord blood hematopoietic progenitor cells.

Umbilical cord blood (CB) has been identified as a potential source of hematopoietic stem cells suitable for clinical transplantation. We used long-term cord blood cultures (LTCBC) to evaluate the hematopoietic potential of populations of umbilical CB cells phenotypically defined and isolated by flow cytometry. LTCBC initiated with CD34+HLA-DR+ and CD34+HLA-DR- CB cells were examined over a period of 8 weeks for the production of assayable burst-forming units-erythroid (BFU-E), colony-forming units-granulocyte/macrophage (CFU-GM), and colony-forming units-mixed (CFU-GEMM) in response to repeated additions of stem cell factor (SCF), interleukin-3 (IL-3), IL-6, and either erythropoietin (Epo) or granulocyte-macrophage colony-stimulating factor (GM-CSF). The LTCBC-initiating cell (LTCBC-IC) appeared to be present among CD34+HLA-DR+ cells, in contrast to our previous findings in adult bone marrow (BM), where the long-term culture initiating cells were shown to be CD34+HLA-DR-. In addition, production of BFU-E, CFU-GM, and CFU-GEMM in CB CD34+HLA-DR+ cells displaying low uptake of the supravital dye rhodamine 123 (Rh123) exceeded those detected in the fraction of cells with high uptake of Rh123. Furthermore, on day 21 of LTCBC, the production of the high proliferative potential colony-forming units (HPP-CFC) by CB CD34+HLA-DR+Rh123dull cells was five-fold greater than that detected in cultures initiated with their Rh123bright counterparts. Collectively, these data show that, contrary to what has been documented in adult human BM, LTCBC-IC and presumably CB cells capable of in vivo engraftment reside in the CD34+HLA-DR+Rh123dull fraction of CB. Although the functional significance of these differences between the in vitro behavior of phenotypically defined populations of CB and BM remains to be determined, these findings constitute an objective parameter with which the suitability of CB for clinical transplantation may be assessed.

Retrovirally mediated gene transfer of Arg22 and Tyr22 forms of dihydrofolate reductase into the hematopoietic cell line K562: a comparison of methotrexate resistance.

Mutations in the enzyme dihydrofolate reductase (DHFR) can confer resistance to the inhibitory effects of folate analogs such as methotrexate (Mtx) and trimetrexate (Ttx). Retroviral vectors expressing the DHFR-Arg22 mutants and the newly described DHFR-Tyr22 mutant were used to transduce the hematopoietic cell line K562. In vitro selection of vector-containing cells was documented via polymerase chain reaction and Southern analysis. When proliferation of selected vector-containing cells was evaluated over a range of Mtx concentrations (0.01 to 10 micromol/L), both Arg22 and Tyr22 provided protection from Mtx, but Tyr22 proved superior to Arg22 in conferring Mtx resistance at low concentrations. Ttx proved to be 10- to 100-fold more potent than Mtx in inhibiting proliferation of nontransduced K562, but the relative effectiveness of individual mutants in conferring drug resistance was similar to that of Mtx. Decreasing the amount of folate in the culture medium to more physiological concentrations increased the potency of administered Mtx and Ttx. Drug resistance in retrovirally transduced K562 cells is consistent with the enzymatic characteristics of the individual mutants. Our findings suggest that the new Tyr22 form of DHFR may prove better in conferring drug resistance than the previously reported Arg22 mutant.

Phenotypic analysis of early hematopoietic progenitors in cord blood and determination of their correlation with clonogenic progenitors: relevance to cord blood stem cell transplantation.

Human cord blood (CB) can be utilized as a source of transplantable stem cells. In the clinical setting, the numbers of clonogenic progenitors and nucleated cells are used as indirect measures of the stem cell content of CB samples. In an attempt to define other parameters of engraftment potential, we have determined the numbers of CD34+ cells, and of CD34+ cells co-expressing CD38 and/or HLA-DR in 17 CB samples. At the same time, clonogenic progenitors were assayed in the presence of erythropoietin and different combinations of rhIL-3, rhG-CSF, rhGM-CSF, rh-Steel Factor and medium conditioned by the PU34 primate cell line. Unseparated CB contained a mean of 14.8 x 10(4) CD34+ cells/mL. CD34+CD38-, CD34+DR+ and CD34+DR+ CD38- cells represented 12.2%, 91% and 10% of all CD34+ cells, respectively. Linear regression analysis demonstrated that the total number of CD34+ cells, as well as the different subsets, correlated positively with the numbers of total colonies and day 14 CFU-GM. Furthermore, the proportion of CD34+CD38- cells was significantly higher than has been reported for adult marrow. Cord blood CD34+CD38- and CD34+DR+ cells have a high proliferative potential and are enriched for primitive hematopoietic progenitors. Thus, we conclude that a single collection of CB could be sufficient to engraft an adult recipient, and that quantitation of CD34+ cells and their subsets may be useful in determining the engraftment potential of CB samples.

Preferential sequestration in vitro of BCR/ABL negative hematopoietic progenitor cells among cytokine nonresponsive CML marrow CD34+ cells.

It is believed that long-term cultures of CML marrow cells favor the outgrowth of BCR/ABL negative hematopoietic progenitor cells (HPC) and that this phenomenon may be enhanced with negative hematopoietic regulators which can maintain primitive HPC in a quiescent state. Proliferation of CML marrow CD34+ cells in primary short-term cultures, maintained in the presence or absence of macrophage inhibitory protein-1 alpha (MIP-1 alpha), was tracked with the membrane dye PKH2. After 7 to 10 days it was possible to distinguish between cytokine responsive (CR) CD34+ cells (cells which had divided thus becoming PKH2dim) and cytokine nonresponsive (CNR) CD34+ cells (cells which had not divided and had therefore remained PKH2bright). CR and CNR CD34+ cells were isolated by flow cytometric cell sorting, seeded in secondary long-term cultures, and their progeny cells assayed weekly for their clonogenic progenitor cell content and expression of BCR/ABL by reverse transcriptase polymerase chain reaction (RT-PCR). Whereas CNR cells isolated from control primary cultures (control/CNR) sustained in vitro hematopoiesis, similar cells from cultures treated with MIP-1 alpha (MIP-1 alpha/CNR) supported a higher and, in some patients, a more extended production of clonogenic HPC, indicating that MIP-1 alpha was able to maintain primitive HPC in a quiescent state. Predominance of BCR/ABL negative progenitors in vitro was more evident in secondary cultures initiated with CNR cells than in those initiated with CR cells, especially those established with MIP-1 alpha/CNR cells. Of interest is the observed decline in the percentage of BCR/ABL+ progenitors in these cultures with time. Whereas up to 100% of progenitors were BCR/ABL+ on day 0, by day 14, only 46% of progenitors in MIP-1 alpha/CNR secondary cultures were BCR/ABL+ and by day 28 and beyond, the percentage of BCR/ABL+ progenitors dropped to below 20%. These results suggest that the quiescent nature of normal HPC present in CML marrow may favor their identification via cell tracking and, subsequently, their isolation from the more actively cycling leukemic cells. These studies also confirm the feasibility of employing negative hematopoietic regulators to augment the sequestration of normal HPC among the cytokine nonresponsive fraction of CD34+ cells, an approach that may be clinically feasible for autotransplantation.

Autologous transplantation of mobilized peripheral blood CD34+ cells selected by immunomagnetic procedures in patients with multiple myeloma.

In the use of autologous PBPC transplantation in patients with multiple myeloma, contamination of PBPC with myeloma cells is commonly observed. Enrichment for CD34+ cells has been employed as a method of reducing this contamination. In this study the reduction of myeloma cells in PBPC was accomplished by the positive selection of CD34+ cells using immunomagnetic bead separation (Isolex 300 system). PBPC were mobilized from 18 patients using cyclophosphamide (4.5 g/m2) and G-CSF (10 microg/kg/day). A median of two leukaphereses and one selection was performed per patient. The median number of mononuclear cells processed was 3.50 x 10(10) with a recovery of 1.11 x 10(8) cells after selection. The median recovery of CD34+ cells was 48% (range 17-78) and purity was 90% (29-99). The median log depletion of CD19+ cells was 3.0. IgH rearrangement, assessed by PCR, was undetectable in 13 of 24 evaluable CD34+ enriched products. Patients received 200 mg/m2 of melphalan followed by the infusion of a median of 2.91 x 10(6)/kg CD34+ cells (1.00-16.30). The median time to absolute neutrophil count >0.5 x 10(9)/l was 11 days, and sustained platelet recovery of >20 x 10(9)/l was 14 days. We conclude that immunomagnetic-based enrichment of CD34+ cells results in a marked reduction in myeloma cells without affecting engraftment kinetics.

Rapid engraftment after allogeneic transplantation using CD34-enriched marrow cells.

Bone marrow cells expressing the surface antigen CD34 comprise approximately 1% of harvested marrow and are highly enriched for marrow progenitor cells, including the cells believed to be responsible for long-term engraftment following bone marrow transplantation (BMT). Selection of CD34-expressing cells was applied in allogeneic BMT (alloBMT) to decrease the number of T lymphocytes in the infused marrow in an attempt to prevent severe graft-versus-host disease (GVHD). We report 14 patients who underwent HLA-identical sibling-matched alloBMT with marrow-enriched for CD34 cells using the Isolex 300 SA device. Patients received total body irradiation, thiotepa, cyclophosphamide, antithymocyte globulin and methylprednisolone prior to marrow infusion. No post-transplantation immunosuppressive therapy was given except for a 5-week course of steroids. The purity of the infused marrow was 64.9+/-6.0% (mean +/- s.e.m.) CD34-positive cells and patients received a mean of 1.24+/-0.21 x 10(6) CD34 cells/kg. A mean of 9.4+/-1.7 x 10(4) CD3 T cells/kg were present in the CD34-enriched product, representing a 2.7+/-0.1 log depletion. There were no graft rejections and patients achieved a sustained absolute granulocyte count of >500 in a median of 10.5 days and a sustained platelet engraftment of >20000 untransfused in a median of 27 days. Patients were discharged a median of 21.5 days after marrow infusion. There were no instances of grade III or IV graft-versus-host disease (GVHD) and no unexpected adverse events during the transplant hospitalization. With a median follow-up of 12 months, the estimated 100 day survival is 86+/-9%. CD34 selection in alloBMT permits rapid engraftment without unanticipated toxicities.

Proliferation of human hematopoietic bone marrow cells in simulated microgravity.

Expansion and/or maintenance of hematopoietic stem cell (HSC) potential following in vitro culture remains a major obstacle in stem cell biology and bone marrow (BM) transplantation. Several studies suggest that culture of mammalian cells in microgravity (micro-g) may reduce proliferation and differentiation of these cells. We investigated the application of these findings to the field of stem cell biology in the hopes of expanding HSC with minimal loss of hematopoietic function. To this end, BM CD34+ cells were cultured for 4-6 d in rotating wall vessels for simulation of micro-g, and assessed for expansion, cell cycle activation, apoptosis, and hematopoietic potential. While CD34+ cells cultured in normal gravity (1-g) proliferated up to threefold by day 4-6, cells cultured in micro-g did not increase in number. As a possible explanation for this, cells cultured in simulated micro-g were found to exit G0/G1 phase of cell cycle at a slower rate than 1-g controls. When assayed for primitive hematopoietic potential in secondary conventional 1-g long-term cultures, cells from initial micro-g cultures produced greater numbers of cells and progenitors, and for a longer period of time, than cultures initiated with 1-g control cells. Similar low levels of apoptosis and adhesion molecule phenotype in micro-g and 1-g-cultured cells suggested similar growth patterns in the two settings. These data begin to elucidate the effects of micro-g on proliferation of human hematopoietic cells and may be potentially beneficial to the fields of stem cell biology and somatic gene therapy.

Evaluation of ex vivo expansion potential of cord blood and bone marrow hematopoietic progenitor cells using cell tracking and limiting dilution analysis.

In the absence of conclusive assays capable of determining the functionality of ex vivo expanded human hematopoietic progenitor cells, we combined cell tracking with the membrane dye PKH2, immunostaining for CD34, and limiting dilution analysis to estimate the frequency of long-term hematopoietic culture-initiating cells (LTHC-ICs) among de novo-generated CD34+ cells. Umbilical cord blood (CB) and bone marrow (BM) CD34+ cells were stained with PKH2 on day 0 and cultured with stem cell factor (SCF) and interleukin-3 (IL-3) in short-term stromal cell-free suspension cultures. Proliferation of CD34+ cells in culture was tracked through their PKH2 fluorescence relative to day 0 and the continued expression of CD34. As such, it was possible to identify cells that had divided while maintaining the expression of CD34 (CD34+PKH2dim) and others that expressed CD34 but had not divided (CD34+PKH2bright). In all such cultures, a fraction of both BM and CB CD34+ cells failed to divide in response to cytokines and persisted in culture for up to 10 days as CD34+PKH2bright cells. Between days 5 and 7 of culture, CD34+PKH2bright and CD34+PKH2dim cells were sorted in a limiting dilution scheme into 96-well plates prepared with medium, SCF, IL-3, IL-6, granulocyte-macrophage colony-stimulating factor, and erythropoietin. Cells proliferating in individual wells were assayed 2 weeks later for their content of clonogenic progenitors and the percentage of negative wells was used to calculate the frequency of LTHC-ICs in each population. Among fresh isolated BM and CB CD34+ cells, the frequencies of LTHC-ICs were 2.01% +/- 0.98% (mean +/- SEM) and 7.56% +/- 2.48%, respectively. After 5 to 7 days in culture, 3.00% +/- 0.56% of ex vivo-expanded BM CD34+PKH2bright cells and 4.46% +/- 1.10% of CD34+PKH2dim cells were LTHC-ICs. In contrast, the frequency of LTHC-IC in ex vivo expanded CB CD34+ cells declined drastically, such that only 3.87% +/- 2.06% of PKH2bright and 2.29% +/- 1.75% of PKH2dim cells were determined to be initiating cells after 5 to 7 days in culture. However, when combined with a calculation of the net change in the number of CD34+ cells in culture, the sum total of LTHC-ICs in both BM and CB cells declined in comparison to fresh isolated cells, albeit to a different degree between the two tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Ex vivo expansion of hematopoietic stem and progenitor cells: are we there yet?

Ex vivo expansion of hematopoietic stem and progenitor cells is a very ambitious idea that would have major implications in the areas of stem cell transplantation and somatic gene therapy. However, successful ex vivo expansion has evaded and frustrated scientists for a number of years. The goal of ex vivo expansion is to induce cell division and proliferation of stem cells while maintaining their primary functional characteristic, namely, their ability to engraft and sustain long-term hematopoiesis. Only when a balance between these two requirements is reached can ex vivo expansion of stem cells be considered successful. Establishing such a balance has not been easy. However, many lessons have been learned along the way, and today we have a more profound understanding of the potential obstacles facing ex vivo expansion than we did only a few years ago. In this review, we discuss these obstacles and evaluate the current status of ex vivo expansion of stem and progenitor cells both from the perspective of basic stem cell biology and from the viewpoint of clinical utility of these cells in transplantation.

Chronic myelogenous leukaemia CD34+ cells exit G0/G1 phases of cell cycle more rapidly than normal marrow CD34+ cells.

To investigate the mechanisms behind the leukaemic expansion of chronic myelogenous leukaemia (CML), we examined the cell cycle status and activation kinetics of purified subpopulations of CD34+ cells from normal and CML bone marrow (BM). Propidium iodide staining was used to assess cell cycle status of fresh cells or those stimulated with cytokines. Although the cell cycle status of fresh low-density cells from CML and normal BM was similar, a larger percentage of CML CD34+ cells were cycling than those from normal BM. The HLA-DR compartment of CML CD34+ cells, a fraction enriched for normal, non-leukaemic progenitors, contained a higher percentage of quiescent cells than the CD34+ HLA-DR+ fraction. When the activation of CD34+ cells was examined in response to SCF or IL-3 alone, or SCF+IL-3+IL-6, CML CD34+ cells exited GO/G1 more rapidly than normal CD34+ cells. Interestingly, although normal BM CD34+ cells failed to cycle in response to IL-6 alone, or in the absence of exogenous cytokines, 30% of CML cells cycled under these conditions. No differences in the degree of apoptosis were documented among CML and normal CD34+ cells in these cultures. These data suggest that enhanced cell cycle activation of CML CD34+ cells, by either autocrine stimuli or via enhanced sensitivity to exogenous stimuli, may be partially responsible for the pronounced cellular expansion characteristic of CML.

Human umbilical cord blood hematopoietic progenitor cells: are they the same as their adult bone marrow counterparts?

In an attempt to expand the hematopoietic progenitor cell (HPC) content of a single collection of umbilical cord blood (CB), we investigated the ex vivo proliferative potential of CB CD34+ cells and the rate of exit of these cells from G0/G1 phases of cell cycle in response to different cytokine combinations. Initial experiments in which phenotypically defined populations of CB and adult bone marrow (BM) CD34+ cells were examined for their HPC content revealed that, contrary to BM, CB CD34+ human leukocyte A (HLA)-DR+ cells appeared to contain the majority of primitive HPC. In cultures of BM CD34+ HLA-DR+ cells incubated with stem cell factor (SCF)+interleukin-3 (IL-3), CD34+ cells increased five-fold over 5 days, while CD34+ cells from CB CD34+ HLA-DR+ cultures increased 11-fold under these same conditions, illustrating an enhanced proliferative potential of CB CD34+ HLA-DR+ cells vs. similar cells from adult BM. Furthermore, a 6.2-fold increase in the number of CB CD34+ still residing in G0/G1 was observed on day 5 in cultures supplemented with SCF and IL-3, suggesting the generation of large numbers of primitive HPC in vitro. The effect of SCF on the exit of CB and BM CD34+ HLA-DR+ cells from G0/G1 was then examined. Following 36- to 48-hour exposure to SCF, 45% of quiescent CB cells exited G0/G1 in contrast to only 13% of quiescent BM cells. In serum-free media supplemented with either SCF or IL-3 alone, CB CD34+ HLA-DR+ cells did not exit G0/G1 phases of cell cycle as rapidly as when CB plasma was present, unless SCF and IL-3 were added simultaneously. Collectively, these results suggest that CB CD34+ cells are more responsive to cytokine stimulation, especially SCF, and may represent more suitable candidates for ex vivo expansion of HPC than BM cells. Furthermore, these data illustrate potentially important biologic differences between the HPC content of subpopulations of BM and CB cells, and the response of these subpopulations to cytokine stimulation.

Orderly process of sequential cytokine stimulation is required for activation and maximal proliferation of primitive human bone marrow CD34+ hematopoietic progenitor cells residing in G0.

Bone marrow (BM) CD34+ cells residing in the G0 phase of cell cycle may be the most suited candidates for the examination of cell cycle activation and proliferation of primitive hematopoietic progenitor cells (HPCs). We designed a double simultaneous labeling technique using both DNA and RNA staining with Hoechst 33342 and Pyronin Y, respectively, to isolate CD34+ cells residing in G0(G0CD34+). Using long-term BM cultures and limiting dilution analysis, G0CD34+ cells were found to be enriched for primitive HPCs. In vitro proliferation of G0CD34+ cells in response to sequential cytokine stimulation was examined in a two-step assay. In the first step, cells received a primary stimulation consisting of either stem cell factor (SCF), Flt3-ligand (FL), interleukin-3 (IL-3), or IL-6 for 7 days. In the second step, cells from each group were washed and split into four or more groups, each of which was cultured again for another week with one of the four primary cytokines individually, or in combination. Tracking of progeny cells was accomplished by staining cells with PKH2 on day 0 and with PKH26 on day 7. Overall examination of proliferation patterns over 2 weeks showed that cells could progress into four phases of proliferation. Phase I contained cytokine nonresponsive cells that failed to proliferate. Phase II contained cells dividing up to three times within the first 7 days. Phases III and IV consisted of cells dividing up to five divisions and greater than six divisions, respectively, by the end of the 14-day period. Regardless of the cytokine used for primary stimulation, G0CD34+ cells moved only to phase II by day 7, whereas a substantial percentage of cells incubated with SCF or FL remained in phase I. Cells cultured in SCF or FL for the entire 14-day period did not progress beyond phase III but proliferated into phase IV (with <20% of cells remaining in phases I and II) if IL-3, but not IL-6, was substituted for either cytokine on day 7. G0CD34+ cells incubated with IL-3 for 14 days proliferated the most and progressed into phase IV; however, when SCF was substituted on day 7, cells failed to proliferate into phase IV. Most intriguing was a group of cells, many of which were CD34+, detected in cultures initially stimulated with IL-3, which remained as a distinct population, mostly in G0/G1, unable to progress out of phase II regardless of the nature of the second stimulus received on day 7. A small percentage of these cells expressed cyclin E, suggesting that their proliferation arrest may have been mediated by a cyclin-related disruption in cell cycle. These results suggest that a programmed response to sequential cytokine stimulation may be part of a control mechanism required for maintenance of proliferation of primitive HPCs and that unscheduled stimulation of CD34+ cells residing in G0 may result in disruption of cell-cycle regulation.