Weeks Of Long-term Culture Research Articles

Abstract 194: Increased PKR expression inhibits differentiation of hematopoietic stem cells

Abstract Acute myelogenous leukemia (AML) is characterized by a differentiation block at an early stage of hematopoiesis that results in the accumulation of immature myeloid cells and an impaired production of blood cells. This parallels findings in solid tumors that demonstrate differentiation stage is often associated with tumor behavior and generally an immature tumor is more aggressive than the more differentiated counterpart. Thus, new research designed to more fully understand the mechanisms of cellular differentiation may improve our understanding of neoplastic transformation and identify new therapeutic targets. Recently our laboratory has determined that increased expression and activation of the interferon-inducible, dsRNA-activated kinase (PKR) is associated with shorter remission duration and worse survival for patients with AML. PKR is a key mediator of the cellular response to inflammatory stress that has been found to be upregulated by chronic inflammation and aging. Since we have previously reported that mice expressing a PKR transgene (TgPKR) in hematopoietic cells have a reduced number of bone marrow (BM) Lin-, Sca1+, cKit+ hematopoietic stem/progenitor cells (HSPCs) and develop a dysplastic BM with an increased frequency of BM blasts, we investigated whether PKR may affect HSPC differentiation. Results indicate that BM of TgPKR mice contains a significantly reduced number of multipotent progenitor cells (MPPs) that are more frequently arrested in G0/G1 than wild type (WT) mice. In contrast, BM of PKR knockout mice (PKRKO) have an increased frequency of proliferating MPPs (S+G2/M) but a decreased frequency of both Long-term (LT) and Short-term (ST) HSCs compared to either WT or TgPKR mice. Although LT-HSCs of TgPKR mice are more frequently quiescent in vivo they remain functional in vitro as demonstrated by nearly double the colony forming capacity compared to BM of PKRKO mice after >4 weeks in long term culture initiating cell (LTC-IC) assays. In addition, when differentiation of the myeloid cell line K562 is induced by PMA treatment, cells with reduced PKR expression by siRNA knockdown display an increased rate and extent of megakaryocyte differentiation compared to control cells. These findings suggest that increased PKR expression has a previously unrecognized function to inhibit hematopoietic cell differentiation. This may account for why TgPKR mice accumulate quiescent LT-HSCs and develop BM dysplasia with age. Furthermore, these results suggest that increased PKR expression/activation as observed in high-risk MDS, AML and other hematologic malignancies may contribute to leukemogenesis, at least in part, by blocking hematopoietic cell differentiation. In addition, these new findings may be applicable to solid tumors since increased PKR signaling has also been reported to be associated with a more rapid and aggressive course of tumor progression in breast cancer, melanoma and colon cancer. Citation Format: Richard L. Bennett, Xiaodong Cheng, Michael T. Byrne, Amalin Asous, Mohini Patel, Zeel Patel, W. Stratford May. Increased PKR expression inhibits differentiation of hematopoietic stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 194.

Human liver cell spheroids in extended perfusion bioreactor culture for repeated-dose drug testing

Primary cultures of human hepatocyte spheroids are a promising in vitro model for long-term studies of hepatic metabolism and cytotoxicity. The lack of robust methodologies to culture cell spheroids, as well as a poor characterization of human hepatocyte spheroid architecture and liver-specific functionality, have hampered a widespread adoption of this three-dimensional culture format. In this work, an automated perfusion bioreactor was used to obtain and maintain human hepatocyte spheroids. These spheroids were cultured for 3-4 weeks in serum-free conditions, sustaining their phase I enzyme expression and permitting repeated induction during long culture times; rate of albumin and urea synthesis, as well as phase I and II drug-metabolizing enzyme gene expression and activity of spheroid hepatocyte cultures, presented reproducible profiles, despite basal interdonor variability (n = 3 donors). Immunofluorescence microscopy of human hepatocyte spheroids after 3-4 weeks of long-term culture confirmed the presence of the liver-specific markers, hepatocyte nuclear factor 4α, albumin, cytokeratin 18, and cytochrome P450 3A. Moreover, immunostaining of the atypical protein kinase C apical marker, as well as the excretion of a fluorescent dye, evidenced that these spheroids spontaneously assemble a functional bile canaliculi network, extending from the surface to the interior of the spheroids, after 3-4 weeks of culture. Perfusion bioreactor cultures of primary human hepatocyte spheroids maintain a liver-specific activity and architecture and are thus suitable for drug testing in a long-term, repeated-dose format.

A Novel Strategy for Expanding Primitive Leukemic Cells from Chronic Phase CML Patients by Forced Overexpression of a NUP98-HOXA10 Homeodomain Fusion Gene.

Analysis of the leukemic stem cell compartment in CML patients with chronic phase disease remains a major challenge. This is due to the usually low frequency of these cells in the bone marrow and blood of most patients regardless of the WBC count and the fact that they are typically outnumbered by normal hematopoietic stem cells from which they cannot be currently separated. Moreover, thus far it has not been possible to identify conditions for their selective expansion in vitro or in vivo. To pursue this goal, we have begun to explore the effects of certain HOX gene-containing constructs on primitive chronic phase CML cells based on previous evidence that these genes markedly enhance the expansion of primitive normal murine and human cord blood cell numbers without inducing leukemia. Lineage-negative peripheral blood or bone marrow cells from 3 chronic phase CML patients (with >93%, <20% and <6% Ph+ LTC-ICs by G-banding karyotyping) were pre-stimulated overnight in a medium containing a serum substitute and 100 ng/ml hSteel Factor (SF), 100 ng/ml hFlt3-ligand and 20 ng/ml each of hIL-3, hIL-6 and hG-CSF. Cells were then exposed to a lenti-PGK-GFP virus with or without an upstream MDUS-NUP98-HOXA10 homeodomain (HD) element for 5 hours in the same medium. After removal of the virus, the cells were maintained in culture under the same conditions for 2 more days to allow full expression of the transduced genes. At this point, both cultures contained the same number of total cells, GFP+ cells and clonogenic progenitors (BFU-E + CFU-GM + CFU-GEMM); i.e., 2.2±0.5 x105 vs 2.2±0.6 x105 total cells, 1.0±0.2 x105 vs 1.3±0.3 x105 GFP+ cells, 3.6±1.7 x104 vs 3.4±1.7 x104 total CFCs and 1.7±0.9 x104 vs 2.4±1.3 x104 GFP+ CFCs per 105 starting lin- cells. However, after the 2-day post-transduction, cells had been maintained for 6 weeks in longterm cultures (LTCs) containing murine stromal cells producing hIL-3, hSF and hG-CSF, we noted a markedly higher (4 to 74-fold) output of CFCs from the NUP98-HOXA10HD-transduced cells. Moreover, whereas the proportion of GFP+ CFCs in the 2-day post-transduction cultures was on average only 31% and 48 % for the control and tested cells respectively, this increased to >98% in the 6-week LTCs initiated with cells that were overexpressing NUP98-HOXA10HD but remained constant at 39% in the control LTCs - suggesting a significant growth advantage conferred by the NUP98A10HD transgene. Importantly, RT-PCR genotyping of the colonies in these assays showed the majority of LTC-IC-derived CFCs from the NUP98-HOXA10HD-transduced cells to be BCR-ABL+, indicative of an even greater output of CFCs by the NUP98-HOXA10HD transduced BCR-ABL+ vs normal cells. These results highlight the potential of NUP98-HOXA10HD to selectively expand primitive CML cells isolated directly from chronic phase patients which will facilitate their further investigation and use to screen and validate new therapeutic agents.

Chronic Myeloid Leukemia Stem Cells and Their Differentiated Progeny Display Divergent Drug Sensitivities to Imatinib Mesylate and Interferon-Alpha.

Imatinib has largely replaced interferon-alpha (IFN) as front-line therapy in the treatment of chronic myeloid leukemia (CML) because of its favorable toxicity profile and superior initial response rate. However, recent laboratory data demonstrating that CML stem cells may have limited susceptibility to imatinib brings into question the potential durability of these responses. In contrast, responses to IFN often take place over months or years, but extensive long-term clinical data indicate that they are often long lasting. In order to determine if the differences in clinical response kinetics could be explained by the activity of imatinib and IFN against CML stem cells or their differentiated progeny, we examined the effects of each agent on distinct cellular subsets in vitro. CD34+ CML progenitors were isolated from 4 patients with newly diagnosed chronic phase CML and incubated with IFN (1000U/ml) or imatinib (10μM) for 96 hours followed by long-term liquid culture over 1-3 weeks to assay more primitive CML stem cells. The effects of each drug on CML progenitors was determined by calculating the number of CFU-GM positive for BCR-ABL by FISH and comparing these values to the bcr-abl+ CFU-GM formed by untreated control cells. Initially, the CML CFU-GM recovery immediately following 96 hours of drug treatment was 62.7 ± 8% and 9.7 ± 2% from IFN and imatinib-treated groups, respectively. After 3 weeks of long-term culture, CML CFU-GM recovery was 23.4 ± 2.6% following IFN treatment and 57.3 ± 22% after imatinib. Thus, imatinib was significantly more toxic to committed CML progenitors than primitive CML progenitors responsible for the maintenance of long-term liquid cultures (p = 0.04). Conversely, IFN had significantly greater activity against primitive CML progenitors than committed progenitors (p = 0.05). Although IFN has diverse biological properties, the mechanisms responsible for its antileukemic activity are unknown. Treatment of the human CML cell line KT-1 with IFN resulted in increased expression of the myeloid antigens CD33 and myeloperoxidase as well as the inhibition of clonogenic growth demonstrating that IFN induced terminal differentiation. Furthermore, several groups have shown that myeloid growth factors also induce differentiation of CML cells in vitro and clinically enhance the activity of IFN; accordingly, the addition of GM-CSF (200U/ml) augmented the differentiation of KT-1 cells. Moreover, myeloid growth factors were required for differentiation as neutralizing antibodies against GM-CSF and IL-3 inhibited the activity of IFN. The addition of GM-CSF to IFN produced similar effects on clinically derived CD34+ CML cells. Although imatinib is a potent inhibitor of committed CML progenitors, it is relatively ineffective against more primitive CML stem cells. In contrast, IFN appears to act primarily against CML stem cells by inducing terminal differentiation that is dependent on the activity of myeloid growth factors. Imatinib and IFN have divergent effects on CML progenitors at different stages of maturation that may correlate with clinical response kinetics and durability.

1037. Site-Specific Genomic Integration in Hematopoietic Cells

Methods of gene transfer to hematopoietic stem cells (HSCs) that result in safe stable chromosomal integration and persistent gene expression in vivo are desired. HSC are an attractive target as their modification could provide treatments for many inherited blood diseases such as Severe Combined Immunodeficiency (SCID) and acquired diseases such as infection by Human Immunodeficiency Virus (HIV-1). PhiC31 integrase is a site-specific recombinase that catalyses a specific, unidirectional integration reaction between two distinct DNA sites known as attB and attP. It has been demonstrated previously that a plasmid expressing the integrase can mediate the integration of a co-delivered attB-containing plasmid into mammalian chromosomes at pseudo attP-sites (host sites sharing homology to attP, as recognized by phiC31). Here we delivered the non-viral phiC31 integrase system to achieve site-restricted integration of genes into the genome of hematopoietic cells using the AMAXA nucleoporator. We applied the phiC31system to integrate a MND-driven eGFP reporter gene into the genomes of human hematopoietic cell lines and primary CD34+ progenitor cells. In the K562 cell line, we observed stable expression of eGFP in >60% of cells for over 2 months; in contrast, in control cells not treated with integrase, the level of gene expression had fallen to near background (<1%) by 2 weeks. In purified human cord blood and bone marrow CD34+ progenitor cells, the integrase repeatedly improved marking efficiency when compared to controls. However, to date relatively low levels of cells that express eGFP (1% range) have been observed over 4-6 weeks of long-term culture. A LAM-PCR based method was applied to investigate pseudo attP-sites in the marked hematopoietic cells. Analyses of integration sites in these marked cells indicated that the preferred pseudo attP-sites chosen by the phiC31 integrase in hematopoietic cells may differ from those previously reported in other commonly used human cell lines and primary cells. Integration events into these pseudo attP-sites are directly responsible for the high level of marking seen in these hematopoietic studies. We are currently defining the identity and expression context afforded by hematopoietic-preferred integration sites. These encouraging results of long-term stable expression highlight the potential of this likely safer non-viral method for ameliorating diseases affecting the hematopoietic system.

A SIN lentiviral vector containing PIGA cDNA allows long-term phenotypic correction of CD34+-derived cells from patients with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell (HSC) disorder in which an acquired somatic mutation of the X-linked PIGA gene results in a deficiency in GPI-anchored surface proteins. Clinically, PNH is dominated by a chronic hemolytic anemia, often associated with recurrent nocturnal exacerbations, neutropenia, thrombocytopenia, and thrombotic tendency. Allogenic bone marrow transplantation is the only potentially curative treatment for severe forms of PNH but is associated with a high treatment-related morbidity and mortality. HSC gene therapy could provide a new therapeutic option, especially when an HLA-matched donor is not available. To develop an efficient gene transfer approach, we have designed a new SIN lentiviral vector (TEPW) that contains the PIGA cDNA driven by the human elongation factor 1 alpha promoter, the central DNA flap of HIV-1, and the WPRE cassette. TEPW transduction led to a complete surface expression of the GPI anchor and CD59 in PIGA-deficient cell lines without any selection procedure. Moreover, efficient gene transfer was achieved in bone marrow and mobilized peripheral blood CD34(+) cells derived from two patients with severe PNH disease. This expression was stable during erythroid, myeloid, and megakaryocytic liquid culture differentiation. CD59 surface cell expression was fully restored during 5 weeks of long-term culture.

Stem cell defect in aplastic anemia: reduced long term culture-initiating cells (LTC-IC) in CD34+ cells isolated from aplastic anemia patient bone marrow.

Aplastic anemia is associated with quantitative and functional abnormalities in the hematopoietic stem cell compartment. Currently, one of the most primitive human hematopoietic progenitor cells that can be functionally assayed in vitro is the long-term culture-initiating cell (LTC-IC). This assay identifies primitive cells that are capable of producing colonies after five weeks of long-term culture using a limiting dilution method. Previous investigators have demonstrated a significant reduction in the frequency of LTC-IC in bone marrow mononuclear cells (BMMC) isolated from aplastic anemia patients when compared to normal donors. However, immunosuppression of hematopoiesis is a prevalent feature of aplastic anemia. Therefore, we assayed the frequency of LTC-IC in cells expressing the CD34 antigen isolated from bone marrow, a population highly enriched for LTC-IC, of both aplastic anemia patients that had received immunosuppressive therapies (IST, n=13) and normal donors (n=28), thereby minimizing continued immunosuppression by T-cells and regulation by other autologous CD34- cells. We describe a significant reduction of LTC-IC frequency in purified CD34+ populations from aplastic patients (P>0.0001), thus demonstrating a functional difference between this phenotypically defined cell population from patients and normal donors.

The FBMD-1 stroma cell line secretes a unique moiety which can increase retroviral transduction of lineage-committed and primitive human peripheral blood progenitor cells.

Peripheral blood progenitor cells are a prime target for gene therapy approaches. As recent data point to the relevance of soluble stroma factors for the efficient transduction of progenitor cells, we tested the stroma-conditioned medium (SCM) of the two cell lines FBMD-1 and L88/5 as well as desulfated and O-sulfated heparin (HS dS and HS OS) for their effect on transduction of peripheral blood progenitor cells. We transduced CD34+ cells of nine tumor patients with the retroviral SF-MDR vector containing the human multidrug resistance 1 (MDR1) gene under serum-free conditions on the fibronectin fragment CH-296 with or without SCM. Provirus-specific polymerase chain reaction showed a median 1.6-fold higher integration rate of the transgene into committed progenitor cells for the group with added FBMD-1 SCM (P=.008). This was maintained after 2 (P=.02) and, as a trend, after 5 weeks of stroma-dependent long-term culture. We found a median 1.5-fold increase in rhodamine-123 (Rh-123) exclusion in myeloid lineage-committed progeny cells following transduction in the presence of FBMD-1 SCM (P=.0004). After 2 or 5 weeks of long-term culture, a significantly higher proportion of Rh-123(dull) cells could still be detected in the FBMD-1 SCM transduction group (P=.003 and P=.04, respectively). L88/5 SCM or HS OS or HS dS was not effective as supplement for improving gene transfer. The FBMD-1 stroma cell line appears to secrete a unique moiety, which can increase retroviral transduction of lineage-committed and primitive progenitor cells. The FBMD-1 stroma activity is not attributable to heparan sulfate.

Rapid Differentiation of a Rare Subset of Adult Human Lin−CD34−CD38− Cells Stimulated by Multiple Growth Factors In Vitro

Recently, several reports of lineage-negative (lin−) CD34− cells with in vivo hematopoietic activity have focused interest on the properties and growth factor response characteristics of these cells. We have now identified a combination of 5 growth factors that are necessary and sufficient to stimulate a marked mitogenic and differentiation response by a subset of human lin−CD34−CD38− cells present in normal adult human marrow and granulocyte colony-stimulating factor (G-CSF)–mobilized blood. Less than 0.1% of the cells in highly purified (including doubly sorted) lin−CD34−CD38− cells from these 2 sources formed colonies directly in semisolid medium or generated such cells after 6 weeks in long-term culture. Nevertheless, approximately 1% of the same lin−CD34−CD38− cells were able to proliferate rapidly in serum-free liquid suspension cultures containing human flt-3 ligand, Steel factor, thrombopoietin, interleukin-3 (IL-3), and hyper–IL-6 to produce a net 28- ± 8-fold increase in total cells within 10 days. Of the cells present in these 10-day cultures, 5% ± 2% were CD34+ and 2.5% ± 0.9% were erythroid, granulopoietic, megakaryocytopoietic, or multilineage colony-forming cells (CFC) (13 ± 7 CFC per lin−CD34−CD38− pre-CFC). In contrast to lin−CD34+CD38−cells, this response of lin−CD34−CD38− cells required exposure to all of the 5 growth factors used. Up to 1.7 × 105 lin−CD34− adult marrow cells failed to engraft sublethally irradiated NOD/SCID-β2M−/− mice. These studies demonstrate unique properties of a rare subset of lin−CD34−CD38− cells present in both adult human marrow and mobilized blood samples that allow their rapid proliferation and differentiation in vitro within an overall period of 3 to 4 weeks. The rapidity of this response challenges current concepts about the normal duration and coordinated control of these processes in adults.

Rapid Differentiation of a Rare Subset of Adult Human Lin−CD34−CD38− Cells Stimulated by Multiple Growth Factors In Vitro

AbstractRecently, several reports of lineage-negative (lin−) CD34− cells with in vivo hematopoietic activity have focused interest on the properties and growth factor response characteristics of these cells. We have now identified a combination of 5 growth factors that are necessary and sufficient to stimulate a marked mitogenic and differentiation response by a subset of human lin−CD34−CD38− cells present in normal adult human marrow and granulocyte colony-stimulating factor (G-CSF)–mobilized blood. Less than 0.1% of the cells in highly purified (including doubly sorted) lin−CD34−CD38− cells from these 2 sources formed colonies directly in semisolid medium or generated such cells after 6 weeks in long-term culture. Nevertheless, approximately 1% of the same lin−CD34−CD38− cells were able to proliferate rapidly in serum-free liquid suspension cultures containing human flt-3 ligand, Steel factor, thrombopoietin, interleukin-3 (IL-3), and hyper–IL-6 to produce a net 28- ± 8-fold increase in total cells within 10 days. Of the cells present in these 10-day cultures, 5% ± 2% were CD34+ and 2.5% ± 0.9% were erythroid, granulopoietic, megakaryocytopoietic, or multilineage colony-forming cells (CFC) (13 ± 7 CFC per lin−CD34−CD38− pre-CFC). In contrast to lin−CD34+CD38−cells, this response of lin−CD34−CD38− cells required exposure to all of the 5 growth factors used. Up to 1.7 × 105 lin−CD34− adult marrow cells failed to engraft sublethally irradiated NOD/SCID-β2M−/− mice. These studies demonstrate unique properties of a rare subset of lin−CD34−CD38− cells present in both adult human marrow and mobilized blood samples that allow their rapid proliferation and differentiation in vitro within an overall period of 3 to 4 weeks. The rapidity of this response challenges current concepts about the normal duration and coordinated control of these processes in adults.

Retroviral transfer and long-term expression of the adrenoleukodystrophy gene in human CD34+ cells.

Adrenoleukodystrophy (ALD) is a demyelinating disease of the central nervous system that results from a genetic deficiency of ALDP, an ABC protein involved in the transport of very long-chain fatty acids (VLCFAs). The cloning of the ALD gene and the positive effects of allogeneic bone marrow transplantation support the feasibility of a gene therapy approach. We report the retroviral transfer of the ALD cDNA to peripheral blood and bone marrow CD34+ cells from control donors and ALD patients. Prestimulation of these cells with cytokines, followed by infection with the M48-ALD retroviral vector, resulted in 20% transduction efficiency (4-40%) and expression of the vector-encoded ALDP in 20% of CD34+ cells (7.3-50%). Long-term culture (LTC) of transduced CD34+ cells from two ALD patients showed efficient transduction (24-28%) and stable expression (25-32%) of ALDP in derived clonogenic progenitors at 3 weeks of culture. The expression of ALDP in CFU cells derived from 5 and 6 weeks of LTC confirmed the effective transduction of LTC-initiating cells. Expression of ALDP was observed in CD68+ CFU-derived cells, suggesting that monocyte-macrophages, the target bone marrow cells in ALD, were produced from transduced progenitor cells. VL-CFA content was corrected in LTC and CFU-derived cells in proportion to the percentage of transduced cells, indicating that the vector-encoded ALDP was functional. Although not efficient yet to allow a clinical perspective, these results demonstrate the feasibility of ALD gene transfer into CD34+ cells of ALD patients.

Cryopreserved human bone marrow stroma is fully functional in vitro.

Human-marrow long-term culture (LTC) enables maintenance of both stromal and haemopoietic elements of normal bone marrow (NBM) in vitro for 4-6 months. Stroma-based cultures are critical for quantitation of long-term culture initiating cells (LTC-IC), the most primitive human haemopoietic cells measurable in vitro. Supply of NBM can be sporadic, and up to 3-4 weeks in culture is required for stromal maturity. Stroma availability for experimental purposes can therefore be limited. Efforts to produce transformed human and transfected murine stromal cell lines comparable to NBM stroma have had some success. As an alternative, we investigated cryopreserved NBM and cryopreserved performed stroma. Function of cryopreserved and control fresh NBM stroma was similar when evaluated for up to 12 weeks in LTC. We have also demonstrated that stroma derived from cryopreserved NBM or performed cryopreserved NBM stroma can sustain third-party haemopoiesis as efficiently as fresh NBM stroma in LTC. Batched cryopreserved stroma is a convenient, rapidly available, source of functional stroma which avoids the logistic difficulties and lack of standardization associated with stroma from fresh NBM. This important advance will enhance the use of stroma-based LTC in studies of human haemopoiesis.

Density separation of umbilical cord blood and recovery of hemopoietic progenitor cells: implications for cord blood banking.

Umbilical cord blood (CB) has been evaluated as a potential source of hematopoietic stem cells suitable for clinical use in the transplantation setting. Previous reports have documented a significant loss of progenitor cells by any manipulation other than cryopreservation. We have evaluated the feasibility of fractionating and cryopreserving CB samples with minimal loss of progenitor cells. We have compared various separation procedures based on different density gradients in the attempt to obtain the highest depletion of red blood cells (RBC) while maintaining the highest recovery of progenitor cells. We compared three different densities of Percoll (1.069 g/ml, 1.077 g/ml, 1.084 g/ml), sedimentation over poligeline (Emagel ) and sedimentation over poligeline followed by separation over Ficoll/Hypaque (F/H). Separated samples (n = 25) were analyzed for recovery of CD34+ cells and progenitor cells (CFU-GEMM, BFU-E, CFU-GM). Separation by sedimentation over poligeline followed by F/H allowed the highest depletion of RBC (hematocrit of the final cellular suspension 0.4 +/- 0.1%) while maintaining high recovery of CD34+ cells (85.3 +/- 5.6%) and total recovery for CFU-GEMM, BFU-E and CFU-GM. After cryopreservation, recovery of clonogenic progenitors was 82% for CFU-GEMM, 94% for BFU-E, 82% for CFU-GM and 90% for colony-forming units (CFUs) after five weeks of long-term culture (LTC). We further evaluated the effect of stem cell factor (SCF) on the in vitro growth of hemopoietic progenitors and on replating efficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Retroviral mediated expression of CD18 in normal and deficient human bone marrow progenitor cells.

The CD18 gene encodes the beta subunit of leukocyte integrins, and autosomal recessive deficiency of CD18 in humans causes a life-threatening abnormality of granulocyte migration. A high titer amphotropic retrovirus encoding CD18 was used to infect bone marrow cells from normal and CD18-deficient human donors. Infected cells were maintained in a long-term culture system and analyzed (1) using PCR to detect the provirus in granulocyte or macrophage colonies (CFU-GMs) derived from the culture, (2) using reverse transcription-PCR (RT-PCR) to detect transcripts in the floating cells in the culture, and (3) using immunostaining of the floating cells to analyze expression of human CD18. By both cocultivation and supernatant infection, a significant fraction (10-82%) of the CFU-GMs were positive for the provirus after five weeks in long-term culture, and as high as 10-15% of the floating cells were positive by immunostaining after nine weeks in the long-term culture. In all cases, cocultivation showed higher infection efficiency than supernatant infection. This is the first report of the introduction of human CD18 cDNA into the bone marrow progenitor cells of patients with leukocyte adhesion deficiency.

Detection of breakpoint cluster region- negative and nonclonal hematopoiesis in vitro and in vivo after transplantation of cells selected in cultures of chronic myeloid leukemia marrow

Philadelphia (Ph1) chromosome-positive clonogenic progenitors usually disappear within 4 to 6 weeks in long-term cultures established from the marrow of patients with chronic myeloid leukemia (CML). In contrast, coexisting chromosomally normal hematopoietic cells are relatively well maintained. Thus, even though normal cells are initially undetectable, they may become the predominant population. Recently, we have begun to explore the potential of such cultures as a strategy for preparing CML marrow for autografting, and based on cytogenetic studies of the differential kinetics of Ph1-positive and Ph1-negative clonogenic cells, have chosen a 10-day period in culture to obtain maximal numbers of selectively enriched normal stem cells. Here we present the results of molecular analyses of the cells regenerated in vivo for the initial three CML patients to be treated using this approach by comparison with the differentiated cells generated by continued maintenance of an aliquot of the autograft in vitro (using a slightly modified culture feeding procedure to enhance the production and release of cells into the nonadherent fraction after 4 weeks) for the one patient whose genotype made molecular analysis of clonality status also possible. These analyses showed that cells with a rearranged breakpoint cluster region (BCR) gene were not detectable by Southern blotting in either in vitro or in vivo populations of mature cells that might be assumed to represent the progeny of primitive cells present at the end of the initial 10 days in culture. Production of BCR-negative cells was also shown to be temporally correlated with the appearance of nonclonal hematopoietic cells both in culture and in vivo. These findings provide support for the view that prolonged maintenance of CML marrow cells in long-term culture may allow molecular characterization of both the BCR-genotype and clonality status of cells with in vivo regenerative potential.

Detection of Breakpoint Cluster Region-Negative and Nonclonal Hematopoiesis In Vitro and In Vivo After Transplantation of Cells Selected in Cultures of Chronic Myeloid Leukemia Marrow

Abstract Philadelphia (Ph1) chromosome-positive clonogenic progenitors usually disappear within 4 to 6 weeks in long-term cultures established from the marrow of patients with chronic myeloid leukemia (CML). In contrast, coexisting chromosomally normal hematopoietic cells are relatively well maintained. Thus, even though normal cells are initially undetectable, they may become the predominant population. Recently, we have begun to explore the potential of such cultures as a strategy for preparing CML marrow for autografting, and based on cytogenetic studies of the differential kinetics of Ph1-positive and Ph1-negative clonogenic cells, have chosen a 10-day period in culture to obtain maximal numbers of selectively enriched normal stem cells. Here we present the results of molecular analyses of the cells regenerated in vivo for the initial three CML patients to be treated using this approach by comparison with the differentiated cells generated by continued maintenance of an aliquot of the autograft in vitro (using a slightly modified culture feeding procedure to enhance the production and release of cells into the nonadherent fraction after 4 weeks) for the one patient whose genotype made molecular analysis of clonality status also possible. These analyses showed that cells with a rearranged breakpoint cluster region (BCR) gene were not detectable by Southern blotting in either in vitro or in vivo populations of mature cells that might be assumed to represent the progeny of primitive cells present at the end of the initial 10 days in culture. Production of BCR- negative cells was also shown to be temporally correlated with the appearance of nonclonal hematopoietic cells both in culture and in vivo. These findings provide support for the view that prolonged maintenance of CML marrow cells in long-term culture may allow molecular characterization of both the BCR-genotype and clonality status of cells with in vivo regenerative potential.