Committed T lymphocyte stem cells of rats. Characterization by surface W3/13 antigen and radiosensitivity.

The existence of stem cells committed to the T lymphoid lineage was deduced from studying how rat T and B stem cells differ in their expression of membrane W3/13 antigen and in their susceptibility in vivo to gamma irradiation. Stem cell activity of rat bone marrow and fetal liver was measured in long-term radiation chimeras using B and T cell alloantigenic surface markers to identify the progeny of donor cells. Monoclonal mouse anti-rat thymocyte antibody W3/13 labeled approximately 40% of fetal liver cells and 60-70% of young rat bone marrow cells (40% brightly, 25% dimly). Bright, dim, and negative cells were separated on a fluorescence-activated cell sorter. All B and T lymphoid stem cells in fetal liver were W3/13 bright, as were B lymphoid stem cells in bone marrow. W3/13 dim bone marrow had over half the T cell repopulating activity of unseparated marrow but gave virtually no B cell repopulation. In further experiments, the radiosensitivity of endogenous B and T lymphoid stem cells was determined by exposing host rats to between 4.5 and 10 Gy of gamma irradiation before repopulation with genetically marked marrow. The results depended on whether chimerism was assayed before day 50 or after day 100. At early times, a radioresistant T stem cell was indicated, whose activity waned later. Thus committed T stem cells of rats carry moderate amounts of W3/13 antigen and are more radioresistant but less permanently chimeragenic than the stem cells that regenerate B lymphocytes.

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Hematopoietic Stem Cell Engraftment in Bone Marrow Is Dependent upon Gsα.

Blood ◽

10.1182/blood.v108.11.857.857 ◽

2006 ◽

Vol 108 (11) ◽

pp. 857-857

Author(s):

Gregor B. Adams ◽

Ian R. Alley ◽

Karissa T. Chabner ◽

Ung-il Chung ◽

Emily S. Marsters ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Cells ◽

Fetal Liver ◽

Conditional Knockout ◽

Wild Type ◽

Hematopoietic Stem ◽

Cell Engraftment

Abstract During development, hematopoietic stem cells (HSCs) translocate from the fetal liver to the bone marrow, which remains the site of hematopoiesis throughout adulthood. In the bone marrow the HSCs are located at the endosteal surface, where the osteoblasts are a key component of the stem cell niche. The exogenous signals that specifically direct HSCs to the bone marrow have been thought to include stimulation of the chemokine receptor CXCR4 by its cognate ligand stromal derived factor-1α (SDF-1α or CXCL12). However, experiments in which CXCR4−/− fetal liver hematopoietic cells were transplanted into wild-type hosts demonstrated efficient engraftment of the HSCs in the bone marrow. In addition, treatment of HSCs with inhibitors of Gαi-coupled signaling, which blocks transmigration towards SDF-1αin vitro, does not affect bone marrow homing and engraftment in vivo. Therefore, we examined whether Gsα-coupled mechanisms play a key role in the engraftment of the HSCs in the bone marrow environment. Utilizing an inducible-conditional knockout of Gsα, we found that deletion of the gene in hematopoietic bone marrow cells did not affect their ability to perform in the in vitro primitive CFU-C or LTC-IC assay systems. However, Gsα−/− cells were unable to establish effective hematopoiesis in the bone marrow microenvironment in vivo in a competitive repopulation assay (41.1% contribution from wild-type cells versus 1.4% from knockout cells). These effects were not due to an inability of the cells to function in the bone marrow in vivo as deletion of Gsα following establishment of hematopoiesis had no effects on the HSCs. Examining the ability of the HSCs to home to the bone marrow, though, demonstrated that deletion of Gsα resulted in a marked impairment of the ability of the stem cells to localize to the marrow space (approximately 9-fold reduction in the level of primitive cell homing). Furthermore, treatment of BM MNCs with an activator of Gsα augmented the cells homing and thus engraftment potential. These studies demonstrate that Gsα is critical to the localization of HSCs to the bone marrow. Which receptors utilize this pathway in this context remains unknown. However, Gsα represents a previously unrecognized signaling pathway for homing and engraftment of HSCs to bone marrow. Pharmacologic activation of Gsα in HSC ex vivo prior to transplantation offers a potential method for enhancing stem cell engraftment efficiency.

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Hepatic Leukemia Factor Is An Important Regulator Of Hematopoietic Stem Cell Activity and Identity

Blood ◽

10.1182/blood.v122.21.2410.2410 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2410-2410

Author(s):

Karolina Komorowska ◽

Hanna K.A Mikkola ◽

Jonas Larsson ◽

Mattias Magnusson

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Fetal Liver ◽

Cell Activity ◽

Hematopoietic Stem ◽

Fold Reduction ◽

Stem Cell Activity

Abstract The transcription factor Hepatic Leukemia Factor (HLF) was originally identified in a chromosomal translocation with the gene E2A causing a subset of childhood B-lineage acute lymphoid leukemia. Moreover, HLF has been described as a regulator of circadian rhythm and recent findings have implicated HLF as a candidate “stemness” gene in both normal and malignant stem cells. Accordingly, overexpression of HLF in human hematopoietic stem cells (HSC) results in an enhanced reconstitution capability in NOD-SCID mice. However, little is known about HLF’s physiological role in hematopoiesis and HSC regulation. Using quantitative PCR, we found that HLF is highly expressed in mouse (C57Bl/6) HSC and is downregulated upon differentiation (HSC 3.2 (±0.95) fold (p<0.001), LSK 1.9 (±0.47) fold (p<0.05), CMP, GMP MEP all less then 0.1 fold, all values are compared to HPRT). This encouraged us to further investigate HSC function in the absence of HLF. The conventional HLF knockout (KO) mice (C57bl/6 background) were viable, born at normal Mendelian ratios and showed normal hematopoietic parameters (bone marrow cellularity: WT 2.7x107 (±5.4 x106), KO 3.3x107 (±6.4 x106), p>0.2 n=9). In addition, the HLF KO mice demonstrated normal lineage distribution of both mature cells in the peripheral blood and bone marrow as well as the frequency of immunophenotypic HSC (Lin-Sca1+ckit+CD34-Flt3-: WT 0.0005 (±0.5x10-4)%, KO 0.0005 (±0.1x10-3)%; n>10). However, in a serial competitive transplantation assay using whole bone marrow (200 000 cells 1:1 ratio), HLF KO cells demonstrated a significant reduction in reconstitution capacity in primary recipients (WT 56 (±15)%, KO 40.2 (±16)%, p=0.028, n>10), which was further increased in the secondary recipients (WT 87.2 (±26)%, KO 8.7 (±5.8)%, p<0.001, n>10). Almost no engraftment was detected from the HLF KO cells in tertiary recipients. To further evaluate stem cell activity in the absence of HLF, we next enumerated the number of competitive repopulating units (CRU) by limiting dilution assay, which revealed a 2.6 fold reduction, of CRU in the HLF KO mice compared to WT controls (WT 1.6 (±0.4)/105 bone marrow cells, KO 0.6 (±0.2)/105 bone marrow cells). Similarly, transplantation of sorted HSC (Lin-Sca1+ckit+CD34-Flt3-) also showed a 2.4 fold (WT 47.3 (±24)%, KO 19.4 (±25)%, p=0.16, n=9) reduced engraftment of total cells but with enhanced T cell frequency in peripheral blood (WT 19.5 (±6.2)%, KO 40.8 (±7.4)%, p=0.01, n=9). Since we also found that HLF was highly expressed in fetal liver derived HSC, we transplanted fetal liver HLF KO cells from E14.5 in a competitive repopulation setting. In line with the phenotype seen in the adult HLF KO mice, the fetal liver HLF KO cells demonstrated impaired reconstitution ability (WT 52.8 (±16)%, KO 0.9 (±1.4)%, n>10). Intriguingly, the phenotype was stronger than in the adult HLF KO HSC, indicating that HLF is particularly important during the expansion phase of HSC in embryonic development. The underlying mechanism of the reduced HSC activity is still unclear, but preliminary findings show that HLF KO HSC have enhanced ROS levels (WT 337 (±33), KO 510 (±55), p<0.05, n=3) and increased cycling HSC (G0: WT 66.5 (±6.4)%, KO 58.5 (±4.7)%; G1/S/G2/M: WT 33.6 (±6.6)%, KO 41.7 (±4.9)%, n=3). We are currently performing global gene expression analysis to further understand the mechanism of HLF in HSC regulation. Interestingly, we also found that HLF appears to regulate the identity of HSC by modulating the expression of the SLAM code on the cell surface of the HLF KO HSC. In contrast to the normal frequency of LSK Flt3-CD34- cells, the HLF KO mice displayed a 3.5 fold reduction in the frequency of LSK CD150+CD48- cells (WT 1.94x10-4 (±4.4x10-5)%, KO 0.56x10-4 (±1.5x10-5)%, p<0.001 n>10). Strikingly, transplantation of as many as 150 LSK CD150+CD48-HLF KO cells showed a complete lack of repopulating capacity in vivo. This did not correlate to the number of functional HSC seen when transplanting whole bone marrow and indicates that HLF affects the identity of HSC by modulating the expression of the SLAM markers. Taken together, we show here for the first time that HLF has a fundamental role in HSC biology during both fetal and adult hematopoiesis by regulating HSC activity and identity. Disclosures: No relevant conflicts of interest to declare.

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Co-Stimulatory Blockade With CTLA4-Ig Permits Transplantation Of Human Hematopoietic Stem Cells and HLA Incompatible T Cells In NOD/SCID γ Null (NSG) Mouse Model

Blood ◽

10.1182/blood.v122.21.1999.1999 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1999-1999

Author(s):

Annie L. Oh ◽

Dolores Mahmud ◽

Benedetta Nicolini ◽

Nadim Mahmud ◽

Elisa Bonetti ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

T Cells ◽

Stem Cell ◽

T Cell ◽

Nsg Mice ◽

Human Cd34 ◽

Cd34 Cells

Abstract Our previous studies have shown the ability of human CD34+ cells to stimulate T cell alloproliferative responses in-vitro. Here, we investigated anti-CD34 T cell alloreactivity in-vivo by co-transplanting human CD34+ cells and allogeneic T cells of an incompatible individual into NSG mice. Human CD34+ cells (2x105/animal) were transplanted with allogeneic T cells at different ratios ranging from 1:50 to 1:0.5, or without T cells as a control. No xenogeneic GVHD was detected at 1:1 CD34:T cell ratio. Engraftment of human CD45+ (huCD45+) cells in mice marrow and spleen was analyzed by flow cytometry. Marrow engraftment of huCD45+ cells at 4 or 8 weeks was significantly decreased in mice transplanted with T cells compared to control mice that did not receive T cells. More importantly, transplantation of T cells at CD34:T cell ratios from 1:50 to 1:0.5 resulted in stem cell rejection since >98% huCD45+ cells detected were CD3+. In mice with stem cell rejection, human T cells had a normal CD4:CD8 ratio and CD4+ cells were mostly CD45RA+. The kinetics of human cell engraftment in the bone marrow and spleen was then analyzed in mice transplanted with CD34+ and allogeneic T cells at 1:1 ratio and sacrificed at 1, 2, or 4 weeks. At 2 weeks post transplant, the bone marrow showed CD34-derived myeloid cells, whereas the spleen showed only allo-T cells. At 4 weeks, all myeloid cells had been rejected and only T cells were detected both in the bone marrow and spleen. Based on our previous in-vitro studies showing that T cell alloreactivity against CD34+ cells is mainly due to B7:CD28 costimulatory activation, we injected the mice with CTLA4-Ig (Abatacept, Bristol Myers Squibb, New York, NY) from d-1 to d+28 post transplantation of CD34+ and allogeneic T cells. Treatment of mice with CTLA4-Ig prevented rejection and allowed CD34+ cells to fully engraft the marrow of NSG mice at 4 weeks with an overall 13± 7% engraftment of huCD45+ marrow cells (n=5) which included: 53±9% CD33+ cells, 22±3% CD14+ monocytes, 7±2% CD1c myeloid dendritic cells, and 4±1% CD34+ cells, while CD19+ B cells were only 3±1% and CD3+ T cells were 0.5±1%. We hypothesize that CTLA4-Ig may induce the apoptotic deletion of alloreactive T cells early in the post transplant period although we could not detect T cells in the spleen as early as 7 or 10 days after transplant. Here we demonstrate that costimulatory blockade with CTLA4-Ig at the time of transplant of human CD34+ cells and incompatible allogeneic T cells can prevent T cell mediated rejection. We also show that the NSG model can be utilized to test immunotherapy strategies aimed at engrafting human stem cells across HLA barriers in-vivo. These results will prompt the design of future clinical trials of CD34+ cell transplantation for patients with severe non-malignant disorders, such as sickle cell anemia, thalassemia, immunodeficiencies or aplastic anemia. Disclosures: No relevant conflicts of interest to declare.

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Demonstration of Thy-1 antigen on pluripotent hemopoietic stem cells in the rat.

Journal of Experimental Medicine ◽

10.1084/jem.148.5.1351 ◽

1978 ◽

Vol 148 (5) ◽

pp. 1351-1366 ◽

Cited By ~ 66

Author(s):

I Goldschneider ◽

L K Gordon ◽

R J Morris

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Cells ◽

Cell Activity ◽

Hemopoietic Stem Cell ◽

Hemopoietic Stem Cells ◽

Stem Cell Activity ◽

Series Of Experiments ◽

Marrow Cells

Three approaches were used to demonstrate the presence of Thy-1 antigen on the surface of pluripotent hemopoietic stem cells in the rat. In the first, stem cells from fetal liver, neonatal spleen, and adult bone marrow were prevented from forming hemopoietic colonies in the spleens of irradiated recipients spleen (colony-forming unit assay) by incubation with antibodies to Thy-1 antigen. Highly specific rabbit heteroantiserum to purified rat brain Thy-1 antigen and mouse alloantisera to Thy-1.1-positive thymocytes were equally effective. This inhibition was neutralized by purified Thy-1 antigen. In a second series of experiments, Thy-1-positive and Thy-1-negative populations of nucleated bone marrow cells were separated by the FACS. All of the hemopoietic stem cell activity was recovered in the Thy-1-positive population. The stem cells were among the most strongly positive for Thy-1 antigen, being in the upper 25th percentile for relative fluorescence intensity. The relationships of Thy-1 antigen to the rat bone marrow lymphocyte antigen (BMLA) was shown in a third series of experiments. Rabbit anti-BMLA serum, which is raised against a null population of lymphocyte-like bone marrow cells, has been shown to have anti-stem cell activity. Here we demonstrate by double immunofluorescence, cocapping, and differential absorption studies that Thy-1 and BMLA are parts of the same molecule.

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Human fetal bone marrow early progenitors for T, B, and myeloid cells are found exclusively in the population expressing high levels of CD34

Blood ◽

10.1182/blood.v84.2.421.bloodjournal842421 ◽

1994 ◽

Vol 84 (2) ◽

pp. 421-432 ◽

Cited By ~ 4

Author(s):

D DiGiusto ◽

S Chen ◽

J Combs ◽

S Webb ◽

R Namikawa ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Cells ◽

Cell Activity ◽

Hematopoietic Stem ◽

Fetal Bone ◽

Cd34 Antigen

Experimentation on human stem cells is hampered by the relative paucity of this population and by the lack of assays identifying multilineage differentiation, particularly along the lymphoid lineages. In our current study, phenotypic analysis of low-density fetal bone marrow cells showed two distinct populations of CD34+ cells: those expressing a high density of CD34 antigen on their surface (CD34hi) and those expressing an intermediate level of CD34 antigen (CD34lo). Multiple tissues were used to characterize the in vitro and in vivo potential of these subsets and showed that only CD34hi cells support long-term B lymphopoiesis and myelopoiesis in vitro and mediate T, B, and myeloid repopulation of human tissues implanted into SCID mice. CD34lo cells repeatedly failed to provide long-term hematopoietic activity in vivo or in vitro. These results indicate that a simple fractionation based on well-defined CD34 antigen levels can be used to reproducibly isolate cells highly enriched for in vivo long-term repopulating activity and for multipotent progenitors, including T- and B-cell precursors. Additionally, given the limited variability in the results and the high correlation between in vitro and in vivo hematopoietic potential, we propose that the CD34hi population contains virtually all of the stem cell activity in fetal bone marrow and therefore is the population of choice for future studies in hematopoietic stem cell development and gene therapy.

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Phenotypic analysis of mouse hematopoietic stem cells shows a Thy-1- negative subset

Blood ◽

10.1182/blood.v80.8.1957.bloodjournal8081957 ◽

1992 ◽

Vol 80 (8) ◽

pp. 1957-1964 ◽

Cited By ~ 2

Author(s):

GJ Spangrude ◽

DM Brooks

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem Cell ◽

Bone Marrow Cells ◽

Cell Activity ◽

Mouse Strains ◽

Hematopoietic Stem ◽

Stem Cell Activity

Mouse hematopoietic stem cells can be identified and enriched from populations of normal bone marrow cells by immunofluorescent labeling of cell surface molecules followed by flow cytometric separation. We show here that the majority of hematopoietic stem cell activity, as defined by long-term competitive repopulation of irradiated animals and by a secondary transplant assay for spleen colony-forming units (CFU- S), could be localized in Ly-6b haplotype mice to a fraction of bone marrow cells that expresses the Ly-6A/E (Sca-1) molecule. Further, an analysis of hematopoietic stem cell activity in bone marrow of mouse strains expressing the Thy-1.1 allele indicated that the vast majority of activity was included in the Thy-1low population. In contrast, hematopoietic stem cell activity found in the bone marrow of Thy-1.2 genotype mouse strains was recovered in both the Thy-1neg and the Thy- 1low populations. However, similar to Thy-1.1 strains, most activity was localized to the Ly-6A/E+ population of cells. The difference in Thy-1 phenotype of hematopoietic stem cell activity apparent between Thy-1.1- and Thy-1.2-expressing mouse strains was not caused by differences in the staining intensity of monoclonal antibodies (MoAbs) specific for the Thy-1 alleles. Furthermore, an antiframework MoAb that stains both alleles of Thy-1 separated hematopoietic stem cell activity from mice expressing the two alleles in the same manner as did allele- specific MoAb. The results of this study show that Thy-1 expression is not an invariant characteristic of mouse hematopoietic stem cells, and that mice expressing the Thy-1.1 allele are unique in that hematopoietic stem cell activity is found exclusively in the Thy-1low population.

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Fluorescence-Based Selection of Gene-Corrected Hematopoietic Stem and Progenitor Cells From Acid Sphingomyelinase-Deficient Mice: Implications for Niemann-Pick Disease Gene Therapy and the Development of Improved Stem Cell Gene Transfer Procedures

Blood ◽

10.1182/blood.v93.1.80 ◽

1999 ◽

Vol 93 (1) ◽

pp. 80-86 ◽

Cited By ~ 17

Author(s):

Shai Erlich ◽

Silvia R.P. Miranda ◽

Jan W.M. Visser ◽

Arie Dagan ◽

Shimon Gatt ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Gene Transfer ◽

Progenitor Cells ◽

Fetal Liver ◽

Acid Sphingomyelinase ◽

Hematopoietic Stem

Abstract The general utility of a novel, fluorescence-based procedure for assessing gene transfer and expression has been demonstrated using hematopoietic stem and progenitor cells. Lineage-depleted hematopoietic cells were isolated from the bone marrow or fetal livers of acid sphingomyelinase–deficient mice, and retrovirally transduced with amphotropic or ecotropic vectors encoding a normal acid sphingomyelinase (ASM) cDNA. Anti–c-Kit antibodies were then used to label stem- and progenitor-enriched cell populations, and the Bodipy fluorescence was analyzed in each group after incubation with a Bodipy-conjugated sphingomyelin. Only cells expressing the functional ASM (ie, transduced) could degrade the sphingomyelin, thereby reducing their Bodipy fluorescence as compared with nontransduced cells. The usefulness of this procedure for the in vitro assessment of gene transfer into hematopoietic stem cells was evaluated, as well as its ability to provide an enrichment of transduced stem cells in vivo. To show the value of this method for in vitro analysis, the effects of retroviral transduction using ecotropic versus amphotropic vectors, various growth factor combinations, and adult bone marrow versus fetal liver stem cells were assessed. The results of these studies confirmed the fact that ecotropic vectors were much more efficient at transducing murine stem cells than amphotropic vectors, and that among the three most commonly used growth factors (stem cell factor [SCF] and interleukins 3 and 6 [IL-3 and IL-6]), SCF had the most significant effect on the transduction of stem cells, whereas IL-6 had the most significant effect on progenitor cells. In addition, it was determined that fetal liver stem cells were only approximately twofold more “transducible” than stem cells from adult bone marrow. Transplantation of Bodipy-selected bone marrow cells into lethally irradiated mice showed that the number of spleen colony-forming units that were positive for the retroviral vector (as determined by polymerase chain reaction) was 76%, as compared with 32% in animals that were transplanted with cells that were nonselected. The methods described within this manuscript are particularly useful for evaluating hematopoietic stem cell gene transfer in vivo because the marker gene used in the procedure (ASM) encodes a naturally occurring mammalian enzyme that has no known adverse effects, and the fluorescent compound used for selection (Bodipy sphingomyelin) is removed from the cells before transplantation.

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Searching for hematopoietic stem cells: evidence that Thy-1.1lo Lin- Sca-1+ cells are the only stem cells in C57BL/Ka-Thy-1.1 bone marrow.

Journal of Experimental Medicine ◽

10.1084/jem.175.1.175 ◽

1992 ◽

Vol 175 (1) ◽

pp. 175-184 ◽

Cited By ~ 250

Author(s):

N Uchida ◽

I L Weissman

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Cell Activity ◽

Hematopoietic Stem ◽

Stem Cell Activity ◽

Marrow Cells

Hematopoietic stem cells (HSCs) are defined in mice by three activities: they must rescue lethally irradiated mice (radioprotection), they must self-renew, and they must restore all blood cell lineages permanently. We initially demonstrated that HSCs were contained in a rare (approximately 0.05%) subset of bone marrow cells with the following surface marker profile: Thy-1.1lo Lin- Sca-1+. These cells were capable of long-term, multi-lineage reconstitution and radioprotection of lethally irradiated mice with an enrichment that mirrors their representation in bone marrow, namely, 1,000-2,000-fold. However, the experiments reported did not exclude the possibility that stem cell activity may also reside in populations that are Thy-1.1-, Sca-1-, or Lin+. In this article stem cell activity was determined by measuring: (a) radioprotection provided by sorted cells; (b) long-term, multi-lineage reconstitution of these surviving mice; and (c) long-term, multi-lineage reconstitution by donor cells when radioprotection is provided by coinjection of congenic host bone marrow cells. Here we demonstrate that HSC activity was detected in Thy-1.1+, Sca-1+, and Lin- fractions, but not Thy-1.1-, Sca-1-, or Lin+ bone marrow cells. We conclude that Thy-1.1lo Lin- Sca-1+ cells comprise the only adult C57BL/Ka-Thy-1.1 mouse bone marrow subset that contains pluripotent HSCs.

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The Sooner the Better: Rapid Transduction Enhances the Engraftment/Selection of Gene Corrected Fanconi Anemia HSC.

Blood ◽

10.1182/blood.v110.11.194.194 ◽

2007 ◽

Vol 110 (11) ◽

pp. 194-194 ◽

Cited By ~ 1

Author(s):

Lars U.W. Muller ◽

Michael Milsom ◽

Chad E. Harris ◽

Jeff Bailey ◽

David A. Williams

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Bone Marrow ◽

Stem Cell ◽

Reporter Gene ◽

Fanconi Anemia ◽

Peripheral Blood ◽

Bone Marrow Cells

Abstract Fanconi anemia (FA) is amenable to genetic correction of hematopoietic stem cells (HSCs). However, as demonstrated in previous clinical gene therapy trials, successful extension of murine studies into human therapies is limited by low numbers of target HSC and poor engraftment of transduced FA HSC (Kelly et al., Mol Ther, 2007). To examine the potential biological consequences/benefits of shortened transduction we used a FA mouse model in which HSC are deficient and prone to excessive loss during in vitro manipulation. We applied a rapid transduction protocol (Mostoslavsky et al., Mol Ther, 2005) utilizing lentiviral vectors and demonstrate that this shortened transduction preserves engraftment of FA HSC to the level of C57BL/6 wt cells. Lin− Sca-1+ c-Kit+ bone marrow cells were isolated from Fanca−/− CD45.2 mice and underwent 4-hr rapid (RT) vs. 96-hr conventional (CT) transduction. An equivalent number of transduced cells were transplanted into lethally irradiated CD45.1 BoyJ mice. Analysis of engraftment chimerism three months post transplantation revealed a significantly higher level of engraftment in animals receiving RT vs. CT cells (90% +/− 14% vs. 26% +/− 31%, respectively, p=<0.01). Rapid transduction also resulted in a significant reduction of engraftment failure (0/36 animals RT vs. 20/36 animals CT). Importantly--emphasizing the FA disease-specific stem cell phenotype, RT vs. CT of C57BL/6 wt cells was associated with no significant difference in engraftment of these cells (93% +/− 1.2% RT vs. 84 +/− 19% CT, p=0.33). Analysis of peripheral blood cells expressing the proviral enhanced green fluorescent protein (eGFP) reporter gene revealed a normal distribution of B-lymphocytes (B220), T-lymphocytes (CD3 epsilon), and granulocytes (MAC-1), indicating multi-lineage engraftment of gene modified cells. In spite of this engraftment advantage, transduction efficiency was low (<30%) using RT. The 6-benzylguanine (6-BG) resistant P140K mutant of O6-methylguanine DNA methyltransferase (MGMTP140K) confers a selective advantage to tranduced HSC treated with alkylating drugs. Following RT with a MGMTP140K/ eGFP expressing lentivirus, 5/6 mice treated with 6-BG and the alkylating drug temozolomide showed a significant rise in the percentage of GFP reporter gene expression in peripheral blood. We extended this approach to the FA model by generating a tri-cistronic lentiviral vector expressing the FANCA cDNA, MGMTP140K, and eGFP. Despite modest in vivo gene marking with this vector, up to 37-fold selection (85% GFP-positive cells) was achieved following exposure of bone marrow of transplant recipients to 6-BG and the alkylating drug temozolomide in vitro. Concurrently, phenotypic correction of mitomycin C hypersensitivity of transduced Fanca−/− bone marrow cells was observed. These data suggest that RT improves stem cell engrafting capacity of FA stem cells in a relevant animal model of stem cell gene therapy. The combination of RT and in vivo selection may allow more successful reconstitution of the lympho-hematopoietic system in gene therapy applications.

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Phenotypic analysis of mouse hematopoietic stem cells shows a Thy-1- negative subset

Blood ◽

10.1182/blood.v80.8.1957.1957 ◽

1992 ◽

Vol 80 (8) ◽

pp. 1957-1964 ◽

Cited By ~ 37

Author(s):

GJ Spangrude ◽

DM Brooks

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem Cell ◽

Bone Marrow Cells ◽

Cell Activity ◽

Mouse Strains ◽

Hematopoietic Stem ◽

Stem Cell Activity

Abstract Mouse hematopoietic stem cells can be identified and enriched from populations of normal bone marrow cells by immunofluorescent labeling of cell surface molecules followed by flow cytometric separation. We show here that the majority of hematopoietic stem cell activity, as defined by long-term competitive repopulation of irradiated animals and by a secondary transplant assay for spleen colony-forming units (CFU- S), could be localized in Ly-6b haplotype mice to a fraction of bone marrow cells that expresses the Ly-6A/E (Sca-1) molecule. Further, an analysis of hematopoietic stem cell activity in bone marrow of mouse strains expressing the Thy-1.1 allele indicated that the vast majority of activity was included in the Thy-1low population. In contrast, hematopoietic stem cell activity found in the bone marrow of Thy-1.2 genotype mouse strains was recovered in both the Thy-1neg and the Thy- 1low populations. However, similar to Thy-1.1 strains, most activity was localized to the Ly-6A/E+ population of cells. The difference in Thy-1 phenotype of hematopoietic stem cell activity apparent between Thy-1.1- and Thy-1.2-expressing mouse strains was not caused by differences in the staining intensity of monoclonal antibodies (MoAbs) specific for the Thy-1 alleles. Furthermore, an antiframework MoAb that stains both alleles of Thy-1 separated hematopoietic stem cell activity from mice expressing the two alleles in the same manner as did allele- specific MoAb. The results of this study show that Thy-1 expression is not an invariant characteristic of mouse hematopoietic stem cells, and that mice expressing the Thy-1.1 allele are unique in that hematopoietic stem cell activity is found exclusively in the Thy-1low population.

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