Wild-type Stem Cells Research Articles

Connexin 31 (GJB3) Deficiency in Mouse Trophoblast Stem Cells Alters Giant Cell Differentiation and Leads to Loss of Oxygen Sensing1

The nonphysiological placental oxidative environment has been implicated in many complications during human pregnancy. Oxygen tension can influence a broad spectrum of molecular changes leading to alterations in trophoblast cell lineage development. In this study, we report that mouse wild-type trophoblast stem cells (TSCs) react to low oxygen (3%) with an enhanced differentiation into the giant cell pathway, indicated by a downregulation of the early stem cell markers Eomes and Cdx2 as well as by a significant upregulation of Tfap2c and the differentiation markers Tpbpa and Prl3d1. Here we demonstrated that connexin 31/GJB3-deficient TSCs failed to stabilize HIF-1A under low oxygen, resulting in nonresponsiveness of different marker genes, such as Cdx2 and Eomes and Tfap2c and Tpbpa. Moreover, connexin 31-deficient TSCs revealed a shift in giant cell differentiation from Prl3d1 expressing parietal giant cells to Ctsq, Prl3b1, and Prl2c2-positive giant cells, probably sinusoidal and canal lining trophoblast giant cells. Thus, loss of connexin 31 led to different giant cell subtypes which bypass the progenitor regulators Tfap2c and Tpbpa under low oxygen conditions.

Myf5 haploinsufficiency reveals distinct cell fate potentials for adult skeletal muscle stem cells

Barbara Gayraud-Morel, Fabrice Chretien*, Aurelie Jory*, Ramkumar Sambasivan*, Elisa Negroni, Patricia Flamant, Guillaume Soubigou, Jean-Yves Coppee, James Di Santo, Ana Cumano, Vincent Mouly and Shahragim Tajbakhsh Stem Cells and Development, Department of Developmental Biology, Institut Pasteur, CNRS URA 2578; Unite d’Histopathologie humaine et modeles animaux; Unite Transcriptome et Epigenome; Unite d’Immunite Innee; Unite du Developpement des Lymphocytes, Institut Pasteur, 25 rue du Dr Roux, 75105 Paris, France Universite Pierre et Marie Curie (Paris 6) UM76, INSERM UMRS974, CNRS UMR7215, Institut de Myologie, Groupe Hospitalier Pitie-Salpetriere, 47-83 bd de l’Hopital, 75651 Paris Cedex 13, France

Myf5 haploinsufficiency reveals distinct cell fate potentials for adult skeletal muscle stem cells

Skeletal muscle stem cell fate in adult mice is regulated by crucial transcription factors, including the determination genes Myf5 and Myod. The precise role of Myf5 in regulating quiescent muscle stem cells has remained elusive. Here we show that most, but not all, quiescent satellite cells express Myf5 protein, but at varying levels, and that resident Myf5 heterozygous muscle stem cells are more primed for myogenic commitment compared with wild-type satellite cells. Paradoxically however, heterotypic transplantation of Myf5 heterozygous cells into regenerating muscles results in higher self-renewal capacity compared with wild-type stem cells, whereas myofibre regenerative capacity is not altered. By contrast, Pax7 haploinsufficiency does not show major modifications by transcriptome analysis. These observations provide a mechanism linking Myf5 levels to muscle stem cell heterogeneity and fate by exposing two distinct and opposing phenotypes associated with Myf5 haploinsufficiency. These findings have important implications for how stem cell fates can be modulated by crucial transcription factors while generating a pool of responsive heterogeneous cells.

Neuroprotection and Behavior Regulation by Adrenomedullin and PAMP as Shown by Conditional Knockout Models

The adrenomedullin (AM) gene, adm, is widely expressed in the central nervous system and several functions have been suggested for brain AM. Until recently, a formal confirmation of these actions using genetic models has been elusive since the systemic adm knockout results in embryo lethality. The recent development of Cre/loxP conditional knockouts for this gene has opened a door for detailed physiopathological studies in the absence of neural AM. These animals present a subtle but very informative phenotype. For instance, it has been shown that neural stem cells lacking adm have delayed growth and their differentiation pattern is different from the one observed in wild type stem cells, producing less neurons and astrocytes but more oligodendrocytes. When the behavior of these animals was studied, it was found that knockout mice were more active, had less motor coordination and were more anxious than the control animals. As it relates to pain sensing, a very complex scenario has emerged; if pure spinal reflexes are tested, then AM functions as a pronociceptive modulator peptide. On the other hand, if the test involves encephalic processing, AM has the opposite effect and acts as an analgesic. Another field where the AM knockouts have shed additional light is in the involvement of AM in neuroprotection. Animals lacking neural AM had larger infarcts when subjected to focal brain ischemia and had compromised survival when exposed to hypobaria. These studies highlight the many functions of AM in the physiopathology of the nervous system.

Abstract 16698: Induction of Mesp1 by Brachyury(t) Generates the Common Multipotent Cardiovascular Stem Cell

Aims: Our recent work demonstrated that common cardiovascular progenitor cells are characterized and induced by the expression of the transcription factor MesP1 in vertebrate embryos and murine embryonic stem cells. As the proliferative potential of stem cell derived cardiomyocytes is limited, it is crucial to understand how MesP1 expression is mediated, in order to achieve reasonable and reliable yields for novel stem cell based therapeutic options. As potential upstream regulators of MesP1 we therefore analysed Eomes and Brachyury(T), which had been controversely discussed to be crucial for cardiovasculogenic lineage formation. Methods and results: Wild-type and transgenic murine embryonic stem cell lines, mRNA-analyses, embryoid body formation, and cell sorting revealed that the MesP1 positive population emerges from the Brachyury(T) positive fraction. In situ hybridizations using wild-type mouse embryos confirmed that Brachyury(T) colocalises with MesP1 in vivo . Likewise, shRNA based loss of Brachyury(T) causes a dramatic decrease of MesP1 expression accompanied by reduced cardiac markers in differentiating embryonic stem cells, which is reflected in vivo via in situs using Brachyury(T) knock-out embryos where MesP1 mRNA is greatly abolished. We finally defined a 3.4 kb proximal MesP1-promoter fragment which is directly bound and activated by Brachyury(T) via a T responsive element as shown via Bandshift, Chromatin Immuneprecipitation and Reporter Assays. Conclusion: Our work contributes to the understanding of the earliest cardiovasculogenic events and may become an important prerequisite for cell therapy, tissue engineering and pharmacological testing in the culture dish using pluripotent stem cell derived as well as directly reprogrammed cardiovascular cell types.

Stem Cell Therapy Extends Incubation and Survival Time in Prion-Infected Mice in a Time Window–Dependant Manner

Prion diseases, which are mostly represented in humans by Creutzfeldt-Jakob disease, are transmissible neurodegenerative disorders characterized by vacuolization and neuronal loss, as well as by the accumulation of an abnormal form of the prion protein. These disorders have yet no effective treatment, and drugs that block prion replication in vitro do not significantly slow down the progression of the disease when used in vivo at late stages. Cell therapy that has been already tested in other neurodegenerative disorders therefore represents an interesting alternative approach. In this study, we showed for the first time in prion diseases that intracerebral transplantation of fetal neural stem cells significantly extended both incubation and survival time. This result was dependant on the time window chosen for the engraftment and was obtained with both genetically modified and wild-type stem cells, therefore forging a path toward efficient stem cell therapy for human prion diseases.

Wnt-inhibitory factor 1 dysregulation of the bone marrow niche exhausts hematopoietic stem cells

AbstractThe role of Wnt signaling in hematopoietic stem cell fate decisions remains controversial. We elected to dysregulate Wnt signaling from the perspective of the stem cell niche by expressing the pan Wnt inhibitor, Wnt inhibitory factor 1 (Wif1), specifically in osteoblasts. Here we report that osteoblastic Wif1 overexpression disrupts stem cell quiescence, leading to a loss of self-renewal potential. Primitive stem and progenitor populations were more proliferative and elevated in bone marrow and spleen, manifesting an impaired ability to maintain a self-renewing stem cell pool. Exhaustion of the stem cell pool was apparent only in the context of systemic stress by chemotherapy or transplantation of wild-type stem cells into irradiated Wif1 hosts. Paradoxically this is mediated, at least in part, by an autocrine induction of canonical Wnt signaling in stem cells on sequestration of Wnts in the environment. Additional signaling pathways are dysregulated in this model, primarily activated Sonic Hedgehog signaling in stem cells as a result of Wif1-induced osteoblastic expression of Sonic Hedgehog. We find that dysregulation of the stem cell niche by overexpression of an individual component impacts other unanticipated regulatory pathways in a combinatorial manner, ultimately disrupting niche mediated stem cell fate decisions.

A patient with a 20-year lag phase between JAK2-V617F+ myeloproliferation and NPM1-mutated AML arguing against a common origin of disease

We have sought to unravel the molecular biology of a female patient who in 1985 at the age of 55 was diagnosed with a chronic myeloproliferative neoplasm (MPN) and in whom overt acute myeloid leukemia (AML) developed in 2005. To this end, DNA and RNA (extracted from either paraffin-embedded bone marrow (BM) or from BM and/or peripheral blood stored in an RNA/DNA-preserving buffer) were analyzed by qPCR and by capillary gel electrophoresis of PCR products. We found the patient to be JAK2-V617F mutation positive throughout the course of disease, while a mutation of the nucleophosmin (NPM1) gene emerged at AML diagnosis and relapse. The 20-yr lag phase between the polycythemia vera and the AML adds indirect evidence to the growing realization that the leukemic transformation in patients with MPN occurs from in a JAK2 wild-type stem cell.

Restoration of home cage activity in Tg2576 mice by immunotherapy with the Ab-oligomer selective antibody A-887755

endogenous neurogenesis occurs, we hypothesized that the neurotrophic factors may improve cognition by increasing endogenous neurogenesis. Therefore, to test this hypothesis, we assessed whether stem cell transplants improved cognition by increasing neurogenesis in the brain. Methods: To test this hypothesis, wild-type neural stem cells derived from Tg2576 X B6SJL/F1 mice were stereotactically injected into the brains of Tg2576 mice (15 mo), followed by BrdU injections. After one month, mice behavior was assessed using various tasks (Morris watermaze, novel object, novel odor task). Mice were subsequently sacrificed. We examined the effect of stem cells on pathology and neurogenesis using stereology. Results: Stem cell transplants in Tg2576 mice rescued cognitive deficits observed in these animals at 15 mo. as assessed with various behavioral tasks. The improvement in cognitive deficits was correlated with neurogenesis in the brain. Conclusions: Here we show that stem cell transplants in AD transgenic mice rescue the cognitive deficits and that this is associated with an increase in neurogenesis.

In Vivo Clonal Analysis Reveals Self-Renewing and Multipotent Adult Neural Stem Cell Characteristics

Neurogenesis and gliogenesis continue in discrete regions of the adult mammalian brain. A fundamental question remains whether cell genesis occurs from distinct lineage-restricted progenitors or from self-renewing and multipotent neural stem cells in the adult brain. Here, we developed a genetic marking strategy for lineage tracing of individual, quiescent, and nestin-expressing radial glia-like (RGL) precursors in the adult mouse dentate gyrus. Clonal analysis identified multiple modes of RGL activation, including asymmetric and symmetric self-renewal. Long-term lineage tracing invivo revealed a significant percentage of clones that contained RGL(s), neurons, and astrocytes, indicating capacity of individual RGLs for both self-renewal and multilineage differentiation. Furthermore, conditional Pten deletion in RGLs initially promotes their activation and symmetric self-renewal but ultimately leads to terminal astrocytic differentiation and RGL depletion in the adult hippocampus. Our study identifies RGLs as self-renewing and multipotent neural stem cells and provides novel insights into invivo properties of adult neural stem cells.

Phosphatidylinositol 3-Kinase (PI3K) Signaling via Glycogen Synthase Kinase-3 (Gsk-3) Regulates DNA Methylation of Imprinted Loci

Glycogen synthase kinase-3 (Gsk-3) isoforms, Gsk-3α and Gsk-3β, are constitutively active, largely inhibitory kinases involved in signal transduction. Underscoring their biological significance, altered Gsk-3 activity has been implicated in diabetes, Alzheimer disease, schizophrenia, and bipolar disorder. Here, we demonstrate that deletion of both Gsk-3α and Gsk-3β in mouse embryonic stem cells results in reduced expression of the de novo DNA methyltransferase Dnmt3a2, causing misexpression of the imprinted genes Igf2, H19, and Igf2r and hypomethylation of their corresponding imprinted control regions. Treatment of wild-type embryonic stem cells and neural stem cells with the Gsk-3 inhibitor, lithium, phenocopies the DNA hypomethylation at these imprinted loci. We show that inhibition of Gsk-3 by phosphatidylinositol 3-kinase (PI3K)-mediated activation of Akt also results in reduced DNA methylation at these imprinted loci. Finally, we find that N-Myc is a potent Gsk-3-dependent regulator of Dnmt3a2 expression. In summary, we have identified a signal transduction pathway that is capable of altering the DNA methylation of imprinted loci.

Crucial function of histone deacetylase 1 for differentiation of teratomas in mice and humans

Histone deacetylase (HDAC) inhibitors induce cell cycle arrest, differentiation or apoptosis in tumour cells and are, therefore, promising anti-cancer reagents. However, the specific HDAC isoforms that mediate these effects are not yet identified. To explore the role of HDAC1 in tumourigenesis and tumour proliferation, we established an experimental teratoma model using wild-type and HDAC1-deficient embryonic stem cells. HDAC1-deficient teratomas showed no significant difference in size compared with wild-type teratomas. Surprisingly, loss of HDAC1 was not only linked to increased apoptosis, but also to significantly enhanced proliferation. Epithelial structures showed reduced differentiation as monitored by Oct3/4 expression and changed E-cadherin localization and displayed up-regulated expression of SNAIL1, a regulator of epithelial cell plasticity. Increased levels of the transcriptional regulator SNAIL1 are crucial for enhanced proliferation and reduced differentiation of HDAC1-deficient teratoma. Importantly, the analysis of human teratomas revealed a similar link between loss of HDAC1 and enhanced tumour malignancy. These findings reveal a novel role for HDAC1 in the control of tumour proliferation and identify HDAC1 as potential marker for benign teratomas.

Shop talk: Annual Drosophila Research Conference, 2010

Shop talk: Annual Drosophila Research Conference, 2010

Effect of disruption of Akt-1 of lin-c-kit+ stem cells on myocardial performance in infarcted heart

We have demonstrated an important role of bone marrow-derived stem cells in preservation of myocardial function. We investigated whether Akt-1 of lin(-)c-kit(+) stem cells preserves ventricular function following myocardial infarction (MI). Isolated lin(-)c-kit(+) cells were conjugated with anti-c-kit heteroconjugated to anti-vascular cell adhesion molecule to facilitate the attachment of stem cells into damaged tissues. Female severe combined immunodeficient mice were used as recipients. MI was created by ligation of the left descending artery. After 48 h, animals were divided into four groups: (i) sham (n = 5): animals underwent thoracotomy without MI; (ii) MI (n = 5): animals underwent MI and received medium; (iii) MI + wild-type (Wt) stem cells (n = 6): MI animals received 5 x 10(5) Wt lin(-)c-kit(+) stem cells; (iv) MI + Akt-1(-/-) stem cells (n = 6): MI animals received 5 x 10(5) Akt-1(-/-) lin(-)c-kit(+) stem cells. Two weeks later, left ventricular function was measured in the Langendorff mode. The peripheral administration of Wt armed stem cells into MI animals restored ventricular function, which was absent in animals receiving Akt-1(-/-) cells. Real-time PCR indicates a decrease in SRY3, a Y chromosome marker in hearts receiving Akt-1(-/-) cells. An increase in angiogenic response was demonstrated in hearts receiving Wt stem cells but not Akt-1(-/-) stem cells. Our results demonstrate that the peripheral administration of Wt lin(-)c-kit(+) stem cells restores ventricular function and promotes angiogenic response following MI. These benefits were abrogated in MI mice receiving Akt-1(-/-) stem cells, suggesting the pivotal role of Akt-1 in mediating stem cells to protect MI hearts.

Resident Macrophages Influence Stem Cell Activity in the Mammary Gland.

Abstract Background: Increasing evidence suggests that macrophages play a role in the normal development of certain organs, including the breast1. Intriguingly, macrophages are often found in the stroma of breast tumours, where they may promote tumour growth and metastasis. The mechanism underpinning the requirement for macrophages during normal development may involve their interactions with mammary stem cells (MaSCs). Recently, a population enriched for MaSCs has been prospectively identified in mice2. We have used 2 approaches to determine whether macrophages are required for normal MaSC function.Methods and Results: The role of macrophages in mammary development was studied in mice with an inactivating recessive mutation in the CSF-1 gene (CSFop/op). MaSCs from CSFop/op mice exhibit significantly reduced clonogenicity in vitro with a reduction in colony size and number in CSFop/op mice. Transplantation assays confirmed a reduced regenerative capacity of the MaSCs derived from CSFop/op mice when transplanted into wild-type recipients. Furthermore, in reciprocal experiments, we found that a macrophage-deficient micro-environment does not support stem cell growth with an inability for wild-type stem cells to repopulate the fat pad of CSFop/op mice.To support these findings, an alternative strategy was used to eliminate the macrophages from the stem cell microenvironment by co-transplanting stem cells with liposomes containing clodronate or saline. Clodronate-containing liposomes are toxic to macrophages after ingestion. There was a greater than 80% impairment in stem cell repopulating frequency as well as a marked decrease in outgrowth potential when cotransplanted with clodronate-containing liposomes.Discussion: These studies indicate a major role for macrophages in supporting stem cell growth during mammary development. This may relate to a role in the stem cell niche. Understanding the mechanisms by which mammary stem cells interact with macrophages during mammary development will shed light on why macrophage infiltration in tumours portends a poor prognosis in breast cancer.1. Gouon-Evans V, Lin EY, Pollard JW. Requirement of macrophages and eosinophils and their cytokines/chemokines for mammary gland development. Breast Cancer Res. 2002; 4:155-64.2. Shackleton M, Vaillant F, Simpson KJ, et al. Generation of a functional mammary gland from a single stem cell. Nature. 2006; 439:84-8. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 49.

A Modified Foamy Viral Envelope Enhances Gene Transfer Efficiency and Reduces Toxicity of Lentiviral FANCA Vectors in Fanca-/- HSCs.

Abstract 696Fanconi anemia (FA) is a recessive DNA repair disorder characterized by bone marrow failure, genomic instability, and a predisposition to malignancies. Stem cell gene transfer technology is a potentially promising therapy, however, we have previously shown that prolonged ex vivo culture of cells using gamma retroviruses, results in a high incidence of apoptosis and predisposes the surviving reinfused cells to hematological malignancy in a murine model of FA. Here, we developed a lentiviral vector encoding the human FANCA cDNA and tested the ability of this construct pseudotyped with either VSV-G or a modified foamy virus (FV) envelope to correct murine Fanca-/- stem and progenitor cells. An overnight transduction protocol was utilized to minimize genotoxic stress due to extended ex vivo manipulations. Transduction and expression of hFANCA was confirmed by three classical functional and biochemical measures in vitro: improved survival of clonogenic progenitors in the presence of mitomycin C (MMC), correction of MMC-induced G2/M arrest, and by the restoration of Fancd2 mono-ubiquitination. Furthermore, in vivo competitive repopulation experiments demonstrated that the repopulating ability of Fanca-/- stem cells transduced with the lentivirus encoding hFANCA was equivalent to that of wild-type stem cells, and the genetically-corrected reconstituting Fanca-/- cells were resistant to MMC and TNF-αa. Importantly, while a significant toxicity was observed using the VSV-G envelope, the toxicity of the FV envelope to murine c-kit+ cells was limited.In parallel experiments, human umbilical cord blood CD34+ cord blood cells were transduced with either a VSV-G- or FV envelope-pseudotyped lentivirus encoding the EGFP reporter gene. Transplantation of 4×105 cells into NOD/SCID/gamma-chainnull yielded a peripheral blood human chimerism comparable to the untransduced control cells (∼30%) regardless of the envelope. However, a much higher gene transfer efficiency of CD34+ cells was observed prior to transplantation when the FV envelope was employed (∼60%), as compared to the VSV-G envelope (∼15%). Furthermore, a similar 4 fold increase in transduction efficiency was observed in peripheral blood and in progenitors isolated from the bone marrow of both primary and secondary long term reconstituted mice. Collectively, these data indicate that the lentiviral construct pseudotyped with FV envelope can efficiently correct murine FA HSC/progenitor cells in a short transduction protocol and that the modified foamy envelope offers significantly greater transduction efficiency at comparable titers in long term reconstituting human cells in a xenograft model. This envelope may offer significant advantages compared to VSV-G in moving forward to phase 1 clinical trials. Disclosures:No relevant conflicts of interest to declare.

Lessons from GNE-deficient embryonic stem cells: sialic acid biosynthesis is involved in proliferation and gene expression

Sialic acids are widely expressed as terminal carbohydrates on glycoconjugates of eukaryotic cells. They are involved in a variety of cellular functions, such as cell adhesion or signal recognition. The key enzyme of sialic acid biosynthesis is the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE), which catalyzes the first two steps of sialic acid biosynthesis in the cytosol. Inactivation of GNE causes early embryonic lethality. In this study, we analyzed wild-type and GNE-deficient embryonic stem cells from mice. We found for the first time that proliferation is directly correlated with GNE-expression and the cellular sialic acid concentration. Furthermore, we identified growth-related genes that are differentially expressed in GNE-deficient embryonic stem cells compared to wild-type embryonic stem cells.

Persistent competition among stem cells and their daughters in theDrosophilaovary germline niche

Cell competition is a short-range cell-cell interaction leading to the proliferation of winner cells at the expense of losers, although either cell type shows normal growth in homotypic environments. Drosophila Myc (dMyc; Dm-FlyBase) is a potent inducer of cell competition in wing epithelia, but its role in the ovary germline stem cell niche is unknown. Here, we show that germline stem cells (GSCs) with relative lower levels of dMyc are replaced by GSCs with higher levels of dMyc. By contrast, dMyc-overexpressing GSCs outcompete wild-type stem cells without affecting total stem cell numbers. We also provide evidence for a naturally occurring cell competition border formed by high dMyc-expressing stem cells and low dMyc-expressing progeny, which may facilitate the concentration of the niche-provided self-renewal factor BMP/Dpp in metabolically active high dMyc stem cells. Genetic manipulations that impose uniform dMyc levels across the germline produce an extended Dpp signaling domain and cause uncoordinated differentiation events. We propose that dMyc-induced competition plays a dual role in regulating optimal stem cell pools and sharp differentiation boundaries, but is potentially harmful in the case of emerging dmyc duplications that facilitate niche occupancy by pre-cancerous stem cells. Moreover, competitive interactions among stem cells may be relevant for the successful application of stem cell therapies in humans.

Mosaic removal of hedgehog signaling in the adult SVZ reveals that the residual wild-type stem cells have a limited capacity for self-renewal.

The Smoothened gene is necessary for cells to transduce hedgehog signaling. Although we and others have previously shown that embryonic removal of Smoothened in the neural tube results in a loss of stem cells from the postnatal subventricular zone, it was unclear whether this reflected a requirement for hedgehog signaling in the establishment or maintenance of the adult niche. Here, we have examined the consequences of conditional removal of Smoothened gene function within the subventricular zone of the adult neural stem cell niche. We observe that both proliferation and neurogenesis are compromised when hedgehog signaling is removed from subventricular zone stem cells. Moreover, even after a 10 month survival period, the stem cell niche fails to recover. It has been reported that the adult subventricular zone quickly rebounds from an antimitotic insult by increasing proliferation and replenishing the niche. During this recovery, it has been reported that hedgehog signaling appears to be upregulated. When mice in which hedgehog signaling in the subventricular zone has been strongly attenuated are given a similar antimitotic treatment, recovery is limited to the reduced level of proliferation and neurogenesis observed before the mitotic insult. Furthermore, the limited recovery that is observed appears to be largely restricted to the minority of neural stem cells that escape the conditional inactivation of Smoothened gene function. These results demonstrate that ongoing hedgehog signaling is required to maintain adult neural stem cells and that their ability to self-renew is limited.

Lentiviral FANCA Vectors Pseudotyped with a Modified Prototype Foamy Viral Envelope Corrects Murine Fanca−/− Hematopoietic Stem Cells with Reduced Toxicity.

Fanconi anemia (FA) is a recessive DNA repair disorder characterized by congenital abnormalities, bone marrow failure, genomic instability, and a predisposition to malignancies. As the majority of FA patients ultimately acquires severe bone marrow failure, transplantation of stem cells from a normal donor is the only curative treatment to replace the malfunctioning hematopoietic system. Stem cell gene transfer technology aimed at re-introducing the missing gene is a potentially promising therapy, however, prolonged ex vivo culture of cells, that was utilized in clinical trials with gammaretroviruses, results in a high incidence of apoptosis and at least in mice predisposes the surviving reinfused cells to hematological malignancy. Consequently, gene delivery systems such as lentiviruses that allow a reduction in ex vivo culture time are highly desirable. Here, we constructed a lentiviral vector expressing the human FANCA cDNA and tested the ability of this construct pseudotyped with either VSVG or a modified prototype foamyvirus (FV) envelope to correct Fanca−/− stem and progenitor cells in vitro and in vivo. In order to minimize genotoxic stress due to extended in vitro manipulations, an overnight transduction protocol was utilized where in the absence of prestimulation, murine Fanca−/− bone marrow cKit+ cells were co-cultured for 16h with FANCA lentivirus on the recombinant fibronectin fragment CH296. Transduction efficiency and transfer of lentivirally expressed FANCA was confirmed functionally in vitro by improved survival of consistently approximately 60% of clonogenic progenitors in serial concentrations of mitomycin C (MMC), irregardless of the envelope that was utilized to package the vector. Transduction of fibroblasts was also associated with complete correction of MMC-induced G2/M arrest and biochemically with the restoration of FancD2 mono-ubiquitination. Finally, to functionally determine whether gene delivery by the recombinant lentivirus during such a short transduction period is sufficient to correct Fanca−/− stem cell repopulation to wild-type levels, competitive repopulation experiments were conducted as previously described. Follow-up of up to 8 months demonstrated that the functional correction were also achieved in the hematopoietic stem cell compartment as evidenced by observations that the repopulating ability of Fanca−/− stem cells transduced with the recombinant lentivirus encoding hFANCA was equivalent to that of wild-type stem cells. Importantly, despite the fact that the gene transfer efficiency into cells surviving the transduction protocol were similar for both pseudotypes, VSVG was associated with a 4-fold higher toxicity to the c-kit+ cells than the FV envelope. Thus, when target cell numbers are limited as stem cells are in FA patients, the foamyviral envelope may facilitate overall greater survival of corrected stem cells. Collectively, these data indicate that the lentiviral construct can efficiently correct FA HSCs and progenitor cells in a short transduction protocol overnight without prestimulation and that the modified foamy envelope may have less cytotoxicity than the commonly used VSVG envelope.