Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential

AbstractFor reasons that are not apparent, it has been difficult to isolate and expand the adult stem cells referred to as mesenchymal stem cells or marrow stromal cells (MSCs) from murine bone marrow. We developed a protocol that provides rapidly expanding MSCs from 5 strains of inbred mice. The MSCs obtained from 5 different strains of mice were similar to human and rat MSCs in that they expanded more rapidly if plated at very low density, formed single-cell–derived colonies, and readily differentiated into either adipocytes, chondrocytes, or mineralizing cells. However, the cells from the 5 strains differed in their media requirements for optimal growth, rates of propagation, and presence of the surface epitopes CD34, stem cell antigen-1 (Sca-1), and vascular cell adhesion molecule 1 (VCAM-1). The protocol should make it possible to undertake a large number of experiments with MSCs in transgenic mice that have previously not been possible. The differences among MSCs from different strains may explain some of the conflicting data recently published on the engraftment of mouse MSCs or other bone marrow cells into nonhematopoietic tissues.

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Enforced P-glycoprotein pump function in murine bone marrow cells results in expansion of side population stem cells in vitro and repopulating cells in vivo

Blood ◽

10.1182/blood.v96.3.902 ◽

2000 ◽

Vol 96 (3) ◽

pp. 902-909 ◽

Cited By ~ 126

Author(s):

Kevin D. Bunting ◽

Sheng Zhou ◽

Taihe Lu ◽

Brian P. Sorrentino

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Cells ◽

Side Population ◽

Murine Bone Marrow ◽

Stem Cell Expansion ◽

Myeloproliferative Syndrome ◽

P Glycoprotein ◽

Marrow Cells

Abstract The human multidrug resistance-1 (MDR1) gene product, P-glycoprotein (P-gp), is well known for its ability to confer drug resistance; however, recent evidence suggests that P-gp expression can have more general effects on cellular development. In support of this idea, it was previously shown that retroviral-mediated MDR1 expression in murine bone marrow cells resulted in the expansion of stem cells in culture and in the development of a myeloproliferative syndrome in transplanted mice. It is now reported that MDR1-mediated stem cell expansion is associated with an increase in side population (SP) stem cells, defined by Hoechst dye staining. Transduction of murine bone marrow cells with an MDR1 retroviral vector resulted in an almost 2 log increase in SP cell numbers over 12 days in culture, whereas there was a rapid loss of SP cells from control cultures. Stem cell amplification was not limited to ex vivo expansion cultures but was also evident when MDR1-transduced cells were directly transplanted into irradiated mice. In these cases, stem cell expansion was associated with relatively high vector copy numbers in stem cell clones. As previously reported, some cases were associated with a characteristic myeloproliferative syndrome. A functionally inactive MDR1 mutant cDNA was used to show that P-gp pump function was required both for amplification of phenotypically defined SP cells and functionally defined repopulating cells. These studies further support the concept that ABC transporter function can have important effects on hematopoietic stem cell development.

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Transplantation of hematopoietic stem cells obtained by a combined dye method fractionation of murine bone marrow

Blood ◽

10.1182/blood.v75.6.1240.1240 ◽

1990 ◽

Vol 75 (6) ◽

pp. 1240-1246 ◽

Cited By ~ 18

Author(s):

I McAlister ◽

NS Wolf ◽

ME Pietrzyk ◽

PS Rabinovitch ◽

G Priestley ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Light Scatter ◽

Isolated Cells ◽

Term Survival ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Separation Media

Abstract Hematopoietic stem cells were purified from murine bone marrow cells (BMC). Their characteristic density, size, internal complexity, Hoechst 33342 dye uptake, and wheat germ agglutinin (WGA) affinity were used to distinguish them from other cells in the bone marrow. BMC suspensions were centrifuged over Ficoll Lymphocyte Separation Media (Organon Teknika, Durham, NC; density 1.077 to 1.08). The lower-density cells were drawn off, stained with Hoechst and labeled with biotinylated WGA bound to streptavidin conjugated to phycoerythrin (WGA-B*A-PE) or with WGA conjugated to Texas Red. These cells were then analyzed and sorted by an Ortho Cytofluorograph 50-H cell sorter. The cells exhibiting medium to high forward light scatter, low to medium right angle light scatter, low Hoechst intensity, and high WGA affinity were selected. Sorted BMC (SBMC) were stained with Romanowsky-type stains for morphologic assay, and were assayed in lethally irradiated (LI) mice for their ability to produce colony-forming units in the spleen (CFU-S) and for their ability to produce survival. The spleen seeding factor for day 8 CFU-S upon retransplantation of the isolated cells was 0.1. The isolated cells were found to have consistent morphology, were enriched up to 135-fold as indicated by day 8 CFU-S assay, 195-fold as indicated by day 14 CFU-S assay, and 150 sorter-selected BMC were able to produce long-term survival in LI mice with retention of donor karyotype. When recipients of this first transplantation were themselves used as BMC donors, their number of day 8 and day 12 CFU-S were found to be reduced. However, 3 X 10(5) of their BMC provided 100% survival among secondary recipients. When the previously SBMC were competed after one transplantation against fresh nonsorted BMC in a mixed donor transplant, they showed the decline in hematopoietic potency normally seen in previously transplanted BMC. We conclude that the use of combinations of vital dyes for fluorescence-activated cell sorting (FACS) selection of survival-promoting murine hematopoietic stem cells provides results comparable with those produced by antibody- selected FACS and has the advantage of a method directly transferable to human BMC.

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Age-Associated Characteristics of Murine Hematopoietic Stem Cells

Journal of Experimental Medicine ◽

10.1084/jem.192.9.1273 ◽

2000 ◽

Vol 192 (9) ◽

pp. 1273-1280 ◽

Cited By ~ 477

Author(s):

Kazuhiro Sudo ◽

Hideo Ema ◽

Yohei Morita ◽

Hiromitsu Nakauchi

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Lymphoid Cells ◽

Differentiation Potential ◽

Self Renewal ◽

Hematopoietic Stem ◽

Functional Changes

Little is known of age-associated functional changes in hematopoietic stem cells (HSCs). We studied aging HSCs at the clonal level by isolating CD34−/lowc-Kit+Sca-1+ lineage marker–negative (CD34−KSL) cells from the bone marrow of C57BL/6 mice. A population of CD34−KSL cells gradually expanded as age increased. Regardless of age, these cells formed in vitro colonies with stem cell factor and interleukin (IL)-3 but not with IL-3 alone. They did not form day 12 colony-forming unit (CFU)-S, indicating that they are primitive cells with myeloid differentiation potential. An in vivo limiting dilution assay revealed that numbers of multilineage repopulating cells increased twofold from 2 to 18 mo of age within a population of CD34−KSL cells as well as among unseparated bone marrow cells. In addition, we detected another compartment of repopulating cells, which differed from HSCs, among CD34−KSL cells of 18-mo-old mice. These repopulating cells showed less differentiation potential toward lymphoid cells but retained self-renewal potential, as suggested by secondary transplantation. We propose that HSCs gradually accumulate with age, accompanied by cells with less lymphoid differentiation potential, as a result of repeated self-renewal of HSCs.

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Implantation of Autologous Bone Marrow Mononuclear Cells in Patients after Acute Myocardial Infarction Via Coronary Artery.

Blood ◽

10.1182/blood.v106.11.4220.4220 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4220-4220

Author(s):

Martin Klabusay ◽

Milan Navratil ◽

Zdenek Koristek ◽

Ladislav Groch ◽

Jaroslav Meluzin ◽

...

Keyword(s):

Myocardial Infarction ◽

Acute Myocardial Infarction ◽

Stem Cells ◽

Bone Marrow ◽

Coronary Artery ◽

Adult Stem Cells ◽

Mononuclear Cells ◽

Cellular Therapy ◽

Bone Marrow Cells ◽

Heart Function

Abstract Background: Several populations of adult stem cells have been identified in bone marrow: hematopoietic stem cells, which are able to regenerate hematopoiesis in all of its lineages, mesenchymal stem cells, which can give rise to connective tissues (bone, cartilage and fat), and endothelial progenitor cells, which can initiate angiogenesis. Adult stem cells are found within the mononuclear cells compartment of bone marrow. Recent reports describe the effect of mononuclear bone marrow cells in reparation of ischemic tissue damage. Methods: The authors designed the experimental protocol of cellular therapy for patients after acute myocardial infarction. Inclusion criteria were: first myocardial infarction treated with primary angioplasty and stent implantation, confirmed non-viability of myocardium by USG, PET and SPECT, elevated CK-MB, and age below 70 years. Patients with intervention on other coronary artery, in unstable condition at day 4 through 6, and with serious non-cardiac disease were excluded. Patients undergoing coronary angioplasty, who signed informed consent, were randomized into three arms: A - high dose of 1•108 mononuclear cells, B - low dose of 1•107 cells, C - no cells. The autologous bone marrow was collected within day 7 after infarction. The mononuclear cells were separated, cultured for 24 hours in serum-free medium, and implanted through catheter via coronary artery into the damaged heart muscle in 7 subsequent injections. Mononuclear cells were analyzed with multicolor flow cytometry and culture assays of CFU-GM and CFU-Meg. The cardiac perfusion, metabolism and function were evaluated with SPECT, PET and echocardiography at 3 months after cell implantation. Results: 31 patients enrolled into the study underwent the protocol (9 in group A, 11 in groups B and C, respectively), and their cardiac functions were evaluated afterwards. There were no serious adverse effects of cell therapy procedure observed in each group. The analysis at 3 months interval showed an improvement in metabolism in the cellular therapy arms detected by PET. Left ventricle ejection fraction improved from 38 to 44%, although this improvement in global heart function was not statistically significant. However, regional heart function at the infarction site (peak systolic velocity of the infarcted wall) improved from 4.1 to 5.0 cm/s in the arm A (p<0.01), while no improvement was observed in arms B and C. A very significant improvement in metabolism and regional function of infarcted area of left ventricle was observed in three patients, all within the treatment arm A. Conclusion: Mononuclear bone marrow cells as a potential source of adult stem cells can be enriched, cultured ex vivo, and safely used in the cellular therapy protocols for ischemic heart disease. The functional benefit of dose of 1•108 mononuclear cells can be detected in a group of patients after acute myocardial infarction.

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Efficient Viral Transduction of Murine Hematopoietic Stem Cells Using HIV-1 Gag-Pol Cross Packaged Simian Immunodeficiency Viral Backbone.

Blood ◽

10.1182/blood.v108.11.5484.5484 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5484-5484

Author(s):

Yuan Lin ◽

Stanton L. Gerson

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Transgene Expression ◽

Bone Marrow Cells ◽

Lentiviral Vector ◽

Bioluminescent Imaging ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Hiv 1 ◽

Marrow Cells

Abstract Lentiviral vectors have been shown to infect non-dividing cells, including hematopoietic stem cell [HSC], and HIV lentiviral vector has been studied extensively in preclinical models. However low HIV lentiviral vector transduction efficiency compared to retroviral vectors, is seen in murine HSC, hampering transplantation and long-term expression of transgene in the recipients. Furthermore, concerns remain regarding the safety of HIV based vectors. Simian Immunodeficiency Viral [SIV] vectors could be safer since the parent virus does not cause disease in humans. However, to model this approach has been difficult because native SIV vectors do not transduce murine cells. We have generated a bicistronic SIV lentiviral SIN vector, containing MGMT and firefly luciferase genes linked by a self-cleavage FMDV 2A sequence. The SIV backbone was kindly provided by Dr. Donald Kohn (University of Southern California). The transgenes are controlled by the MND promoter, which has been shown to express well in murine hematopoietic stem cells. The vector was generated by cross-packaging SIV RNA with HIV-1 ΔR8.91 packaging plasmid and VSVG pseudotyped envelope (Ref. Retrovirology2005, 2:55). Unconcentrated viruses had an average titer of 1E+06 iu/ml, which was similar to HIV-1 lentiviral vectors. In vitro, HIV-1 cross-packaged SIV-mnd-MGMT-2A-Luc vector was able to transduce both human and murine cell lines with no reduction of expression for 10 weeks. In addition, this cross-packaged SIV vector was also able to transduce primary murine bone marrow cells from Balb/C mice with low MOI of 0.5 to 1. Transduced primary murine bone marrow cells maintained transgene expression during a 4 week culture. To analyze in vivo expression, Balb/C bone marrow cells were transduced for 48 hrs in cytokines with the HIV-1 packaged SIV vector and transplanted into irradiated recipients. We used bioluminescent imaging (BLI) to monitor the transgene expression and the dynamic engraftment of transduced murine bone marrow cells. At MOI of 0.5 or 5, transduction efficiencies in murine progenitor cells were 24.4% and 46.7% respectively by PCR of transgene from CFU colonies. Bioluminescent imaging indicated similar engraftment patterns of transduced bone marrow cells by HIV-1 lentiviral vector or cross-packaged SIV lentiviral vector, as early as day 5. Consistent BLI signals indicated sustained expression of transgene in SIV vector transduced bone marrow cells beyond 30 days. With this study, cross-packaged SIV SIN vector could be used as a potential gene transfer vector in both preclinical murine studies and perhaps in clinical trials.

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In Vitro Myelo-Lymphoid Colony Formation by Mouse Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v112.11.3861.3861 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3861-3861

Author(s):

Jun Ooehara ◽

Hina Takano ◽

Shin-ichiro Takayanagi ◽

Hiromitsu Nakauchi ◽

Hideo Ema

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Culture Conditions ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Lymphoid Progenitors ◽

Marrow Cells

Abstract Hematopoietic stem cells (HSCs) clonally differentiate into all myeloid, B-lymphoid, and T-lymphoid lineages. Mouse HSCs are known to form in vitro colonies comprised of morphologically identifiable myeloid cells such as neutrophils, macrophages, erythroblasts, and megakaryocytes. Whether HSCs are able to differentiate along B-and T-lymphoid lineages in such colonies remains obscure. The co-culture systems with stromal cells such as S17, OP9, OP9/Delta cells have been shown to support B- and T-cell development. These systems have been used to identify subclasses of progenitors with lymphoid potentials. However, neither B cells nor T cells have been successfully generated from HSCs in vitro. This is most likely due to the lack of culture conditions which support HSCs to differentiate into a certain stage of lymphoid progenitors. In this study, we attempted to use serum-free single-cell culture to identify cytokines which fill the developmental gap between HSCs and lymphoid progenitors. Here we show that myelo-lymphoid colonies are formed by HSCs in the presence of thrombopoietin (TPO), interleukin (IL)-11, or IL-12 together with stem cell factor (SCF). CD34-negative/low, c-Kit-positive, Sca-1-positive, lineage marker-negative (CD34-KSL) bone marrow cells were individually cultured with a combination of cytokines for 7 days. All cells in each colony were transplanted into each from a group of lethally irradiated mice, along with compromised bone marrow cells. The recipient mice were periodically analyzed after transplantation to detect transient myeloid and lymphoid reconstitution. All myeloid, B-, and T-lymphoid progenitor activities were detected in single colonies formed in the presence of SCF+TPO, SCF+IL-11, SCF+IL-12. Only myeloid progenitor activity was predominantly detected in single colonies formed in the presence of SCF+IL-3, consistent with previous observations in blast colony assays. All these combinations of cytokines support self-renewal in HSCs to varying degrees. We conclude that TPO, IL-11, and IL-12 directly act on HSCs and support them to differentiate into progenitors with lymphoid differentiation potential. Early differentiation pathways in HSCs are likely to be used in common by myeloid and lymphoid lineages and be supported in common by multiple cytokines.

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Enforced P-glycoprotein pump function in murine bone marrow cells results in expansion of side population stem cells in vitro and repopulating cells in vivo

Blood ◽

10.1182/blood.v96.3.902.015k40_902_909 ◽

2000 ◽

Vol 96 (3) ◽

pp. 902-909 ◽

Cited By ~ 4

Author(s):

Kevin D. Bunting ◽

Sheng Zhou ◽

Taihe Lu ◽

Brian P. Sorrentino

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Cells ◽

Side Population ◽

Murine Bone Marrow ◽

Stem Cell Expansion ◽

Myeloproliferative Syndrome ◽

P Glycoprotein ◽

Marrow Cells

The human multidrug resistance-1 (MDR1) gene product, P-glycoprotein (P-gp), is well known for its ability to confer drug resistance; however, recent evidence suggests that P-gp expression can have more general effects on cellular development. In support of this idea, it was previously shown that retroviral-mediated MDR1 expression in murine bone marrow cells resulted in the expansion of stem cells in culture and in the development of a myeloproliferative syndrome in transplanted mice. It is now reported that MDR1-mediated stem cell expansion is associated with an increase in side population (SP) stem cells, defined by Hoechst dye staining. Transduction of murine bone marrow cells with an MDR1 retroviral vector resulted in an almost 2 log increase in SP cell numbers over 12 days in culture, whereas there was a rapid loss of SP cells from control cultures. Stem cell amplification was not limited to ex vivo expansion cultures but was also evident when MDR1-transduced cells were directly transplanted into irradiated mice. In these cases, stem cell expansion was associated with relatively high vector copy numbers in stem cell clones. As previously reported, some cases were associated with a characteristic myeloproliferative syndrome. A functionally inactive MDR1 mutant cDNA was used to show that P-gp pump function was required both for amplification of phenotypically defined SP cells and functionally defined repopulating cells. These studies further support the concept that ABC transporter function can have important effects on hematopoietic stem cell development.

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Constitutively active AKT depletes hematopoietic stem cells and induces leukemia in mice

Blood ◽

10.1182/blood-2009-06-229443 ◽

2010 ◽

Vol 115 (7) ◽

pp. 1406-1415 ◽

Cited By ~ 184

Author(s):

Michael G. Kharas ◽

Rachel Okabe ◽

Jared J. Ganis ◽

Maricel Gozo ◽

Tulasi Khandan ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Leukemic Stem Cells ◽

Myeloproliferative Disease ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Akt Activation ◽

Marrow Cells

Abstract Human cancers, including acute myeloid leukemia (AML), commonly display constitutive phosphoinositide 3-kinase (PI3K) AKT signaling. However, the exact role of AKT activation in leukemia and its effects on hematopoietic stem cells (HSCs) are poorly understood. Several members of the PI3K pathway, phosphatase and tensin homolog (Pten), the forkhead box, subgroup O (FOXO) transcription factors, and TSC1, have demonstrated functions in normal and leukemic stem cells but are rarely mutated in leukemia. We developed an activated allele of AKT1 that models increased signaling in normal and leukemic stem cells. In our murine bone marrow transplantation model using a myristoylated AKT1 (myr-AKT), recipients develop myeloproliferative disease, T-cell lymphoma, or AML. Analysis of the HSCs in myr-AKT mice reveals transient expansion and increased cycling, associated with impaired engraftment. myr-AKT–expressing bone marrow cells are unable to form cobblestones in long-term cocultures. Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) rescues cobblestone formation in myr-AKT–expressing bone marrow cells and increases the survival of myr-AKT mice. This study demonstrates that enhanced AKT activation is an important mechanism of transformation in AML and that HSCs are highly sensitive to excess AKT/mTOR signaling.

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An Improved Harvest andin VitroExpansion Protocol for Murine Bone Marrow-Derived Mesenchymal Stem Cells

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/105940 ◽

2010 ◽

Vol 2010 ◽

pp. 1-10 ◽

Cited By ~ 26

Author(s):

Song Xu ◽

Ann De Becker ◽

Ben Van Camp ◽

Karin Vanderkerken ◽

Ivan Van Riet

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Surface Markers ◽

Differentiation Potential ◽

Murine Bone Marrow ◽

Expansion Time ◽

Expansion Protocol ◽

In Vitro Expansion

Compared to bone marrow (BM) derived mesenchymal stem cells (MSCs) from human origin or from other species, the in vitro expansion and purification of murine MSCs (mMSCs) is much more difficult because of the low MSC yield and the unwanted growth of non-MSCs in the in vitro expansion cultures. We describe a modified protocol to isolate and expand murine BM derived MSCs based on the combination of mechanical crushing and collagenase digestion at the moment of harvest, followed by an immunodepletion step using microbeads coated with CD11b, CD45 and CD34 antibodies. The number of isolated mMSCs as estimated by colony forming unit-fibroblast (CFU-F) assay showed that this modified isolation method could yield 70.0% more primary colonies. After immunodepletion, a homogenous mMSC population could already be obtained after two passages. Immunodepleted mMSCs (ID-mMSCs) are uniformly positive for stem cell antigen-1 (Sca-1), CD90, CD105 and CD73 cell surface markers, but negative for the hematopoietic surface markers CD14, CD34 and CD45. Moreover the immunodepleted cell population exhibits more differentiation potential into adipogenic, osteogenic and chondrogenic lineages. Our data illustrate the development of an efficient and reliable expansion protocol increasing the yield and purity of mMSCs and reducing the overall expansion time.

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High Level Expression of a Membrane-Anchored Inhibitor of HIV Infection in Murine Hematopoietic Stem and Progenitor Cells Does Not Pertubate Serial Multilineage Repopulation.

Blood ◽

10.1182/blood.v104.11.1758.1758 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1758-1758

Author(s):

Axel Schambach ◽

Bernhard Schiedlmeier ◽

Jens Bohne ◽

Dorothee von Laer ◽

Geoff Margison ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Transgene Expression ◽

Bone Marrow Cells ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Hiv 1 ◽

Marrow Cells

Abstract T20 is a 36-amino-acid peptide that binds to HIV-1 gp41 and thereby acts as a fusion inhibitor, thus mediating potent and selective inhibition of HIV-1 entry in vitro and in vivo. An extended peptide expressed as an artificial, membrane-bound molecule (mbC46) efficiently inhibits HIV infection of primary human T-cells following retroviral vector mediated gene transfer (Egelhofer et al., J Virol, 2004). To develop an even more stringent approach to HIV gene therapy, we targeted hematopoietic stem cells. In 3 experimental groups of C57BL/6 mice (9 animals/group), we investigated the long-term toxicity of murine bone marrow cells transduced with M87o, a therapeutic vector designed to coexpress mbC46 and an HIV-derived RNA RRE-decoy to inhibit HIV replication. As controls we used the same vector containing an inactive C46 peptide and mock-transduced cells. Blood samples were collected monthly. Donor chimerism and transgene expression in multiple lineages were determined by FACS analysis and transgene integration was measured by real time PCR. Six months after transplantation, 4 mice per group were sacrificed and the remaining 5 mice per group were observed for another 6 months. In addition to the parameters mentioned above, we performed complete histopathology, blood counts and clinical biochemistry. Donor chimerism in all groups ranged from 82 – 94% (day 190 and day 349). In the M87o group, 60% of donor cells expressed mbC46. FACS data showed persisting transgene expression in T-cells (CD4, CD8, 65%), B-cells (B220, 46%), myeloid cells (CD11b, 68%), platelets (CD41, 19%), and RBC (60%) of the peripheral blood and bone marrow cells. Highly sustained gene marking (2–4 copies/genome) was noticed on day 190. To reveal latent malignant clones potentially originating from side effects of the genetic manipulation, 1x106 bone marrow cells from 4 primary recipients were transplanted into lethally irradiated secondary recipients (3 recipients/primary mouse) and these mice were observed for 8 months. All together, we could not observe any evidence for leukemogenic capacity. Analysis of peripheral blood and bone marrow showed a similar transgene expression pattern compared to the primary mice. To generate a complete chimerism of transgenic cells, we chose the human drug resistance gene methylguanine-methyltransferase (MGMT, P140K) to select for mbC46-transduced stem cells in vitro and in vivo. Different coexpression strategies were tested. Function of the MGMT protein was confirmed in a quantitative alkyltransferase assay and in a cytotoxicity assay using BCNU or temozolomide. In vitro selection of transduced 32D and PM1 cells with benzylguanine and BCNU showed >95% positive cells with evidence of polyclonal survival. Transduced PM1 cells underwent an HIV challenge assay. In vivo experiments in a murine bone marrow transplantation setting are ongoing to determine the potency and safety of combined retroviral expression of mbC46 and MGMT in relevant preclinical models. Successful conclusion of these studies will hopefully result in a phase I clinical trial testing the concept of generating an HIV-resistant autologous hematopoiesis.

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