Lack of self-renewal capacity in Fancc-/- stem cells after ex vivo expansion

Treatments of the hematological manifestation in Fanconi anemia (FA) are first supported by attempts to stimulate hematopoiesis with androgens or hematopoietic growth factors. However, the long-term curative treatment of the hematological manifestation in FA patients is bone marrow (BM) or cord blood stem cell transplantation. The success rate for BM transplantation is fairly high with HLA-matched sibling donors but is, unfortunately, low with HLA-matched unrelated donors. An alternative curative treatment for those patients with no sibling donors might be gene transfer into hematopoietic stem cells. Because FA patients have reduced numbers of stem/progenitor cells, ex vivo expansion of hematopoietic stem cells would be a crucial step in gene transfer protocols. Using the FA mouse model, Fancc-/-, we tested the ability of CD34- hematopoietic stem cells to support ex vivo expansion. We determined that Fancc-/- CD34- stem cells have reduced reconstitution ability and markedly reduced self-renewal ability after culture, as shown by secondary transplants. These results indicate that FA stem cells may not be well suited for ex vivo expansion before gene transfer or transplantation protocols.     

Reduced fertility and hypersensitivity to mitomycin C characterize Fancg/Xrcc9 null mice

Fanconi anemia (FA) is a heterogeneous autosomal recessive chromosomal instability syndrome associated with diverse developmental abnormalities, progressive bone marrow failure and a predisposition to cancer. Spontaneous chromosomal breakage and hypersensitivity to DNA cross-linking agents characterize the cellular FA phenotype. The gene affected in FA complementation group G patients was initially identified as XRCC9, for its ability to partially correct the cellular phenotype of the Chinese hamster ovary (CHO) cell mutant UV40. By targeted disruption we generated Fancg/Xrcc9 null mice. Fancg knock-out (KO) mice were born at expected Mendelian frequencies and showed normal viability. In mice, functional loss of Fancg did not result in developmental abnormalities or a pronounced incidence of malignancies. During a 1 year follow-up, blood cell parameters of Fancg KO mice remained within normal values, revealing no signs of anemia. Male and female mice deficient in Fancg showed hypogonadism and impaired fertility, consistent with the phenotype of FA patients. Mouse embryonic fibroblasts (MEFs) from the KO animals exhibited the FA characteristic cellular response in showing enhanced spontaneous chromosomal instability and a hyper-responsiveness to the clastogenic and antiproliferative effects of the cross-linking agent mitomycin C (MMC). The sensitivity to UV, X-rays and methyl methanesulfonate, reported for the CHO mutant cell line UV40, was not observed in Fancg(-/-) MEFs. Despite a lack of hematopoietic failure in the KO mice, clonogenic survival of bone marrow cells in vitro was strongly reduced in the presence of MMC. The characteristics of the Fancg(-/-) mice closely resemble those reported for Fancc and Fanca null mice, supporting a tight interdependence of the corresponding gene products in a common pathway.     

Fibroblast growth factor-1 and -2 preserve long-term repopulating ability of hematopoietic stem cells in serum-free cultures

In this study, we demonstrate that extended culture of unfractionated mouse bone marrow (BM) cells, in serum-free medium, supplemented only with fibroblast growth factor (FGF)-1, FGF-2, or FGF-1 +2 preserves long-term repopulating hematopoietic stem cells (HSCs). Using competitive repopulation assays, high levels of stem cell activity were detectable at 1, 3, and 5 weeks after initiation of culture. FGFs as single growth factors failed to support cultures of highly purified Lin(-)Sca-1(+)c-Kit(+)(LSK) cells. However, cocultures of purified CD45.1 LSK cells with whole BM CD45.2 cells provided high levels of CD45.1 chimerism after transplant, showing that HSC activity originated from LSK cells. Subsequently, we tested the reconstituting potential of cells cultured in FGF-1 + 2 with the addition of early acting stimulatory molecules, stem cell factor +interleukin-11 + Flt3 ligand. The addition of these growth factors resulted in a strong mitogenic response, inducing rapid differentiation and thereby completely overriding FGF-dependent stem cell conservation. Importantly, although HSC activity is typically rapidly lost after short-term culture in vitro, our current protocol allows us to sustain stem cell repopulation potential for periods up to 5 weeks.     

Hematopoietic repopulating ability of cord blood CD34(+) cells in NOD/Shi-scid mice

Although umbilical cord blood (CB) is increasingly being used as an alternative to bone marrow (BM) as a source of transplantable hematopoietic stem cells (HSC), information on the hematopoietic repopulating ability of CB HSC is still limited. We recently established a xenotransplantation system in NOD/Shi-scid mice to evaluate human stem cell activity. In the present study, we transplanted 5 to 10 x 10(4) CB CD34(+) cells into six NOD/Shi-scid mice treated with anti-asialo GM1 antiserum to investigate the hematopoietic repopulating ability of CB. The BM of all recipients contained human CD45(+) cells 10 to 12 weeks after the transplantation (43.8 +/- 17.7%). Clonal culture of the recipient BM cells revealed the formation of various types of human hematopoietic colonies, including myelocytic, erythroid, megakaryocytic, and multilineage colonies, indicating that CB HSC can differentiate into hematopoietic progenitors of various lineages. However, the extent of the differentiation and maturation differed with each lineage. CD13(+)/CD14(+)/CD33(+) myelocytic cells were mainly repopulated in BM and peripheral blood (PB). While CD41(+) megakaryocytic cells and platelets were present, few glycophorin A(+)CD71(+) or hemoglobin alpha-containing erythroid cells were detected. CD19(+) B cells were the most abundantly repopulated in NOD/Shi-scid mice, but their maturational stage differed among the hematopoietic organs. Most of the BM CD19(+) cells were immature B cells expressing CD10 but not surface immunoglobulin (Ig) M, whereas more mature CD19(+)CD10(-) surface IgM(+) B cells were predominantly present in spleen and PB. CD3(+) T cells were not detected even in the recipient thymus. The transplantation to the NOD/Shi-scid mouse may provide a useful tool for evaluating the repopulating ability of transplantable human HSC.     

Different subsets of haematopoietic cells and immune cells in bone marrow between young and older donors

Young donors are reported to be associated with better transplant outcomes than older donors in allogeneic hematopoietic stem cell transplantation (allo‐HSCT), but the mechanism is still unclear. The current study compared the different subsets of haematopoietic stem cells (HSCs) and their progenitors as well as immune cells in bone marrow (BM) between young and older donors. The frequencies of HSCs, multipotent progenitors (MPPs) and myeloid progenitors, including common myeloid progenitors (CMPs) and megakaryocyte–erythroid progenitors (MEPs), were decreased, whereas those of lymphoid progenitors, including multi‐potent lymphoid progenitors (MLPs) and common lymphoid progenitors (CLPs), were increased in the BM of young donors compared with in that of older donors. Lower reactive oxygen species (ROS) levels were observed in BM HSCs and six progenitor lines in young donors. Furthermore, young donors demonstrated higher frequencies of naive T cells and immune suppressor cells, such as alternative macrophages (M2) and lower frequencies of memory T cells and immune effectors, including T helper‐1 and T cytotoxic‐1 cells, in BM than older donors. Multivariate analysis demonstrated that donor age was independently correlated with BM HSC frequency. Although further validation is required, our results suggest that the differences in the frequency and immune differentiation potential of HSCs in BM between young donors and older donors may partly explain the different outcomes of allo‐HSCT.     

TIS21/(BTG2) negatively regulates estradiol-stimulated expansion of hematopoietic stem cells by derepressing Akt phosphorylation and inhibiting mTOR signal transduction

It has been known that 12-O-tetradecanoyl phorbol-13-acetate-inducible sequence 21 (TIS21), ortholog of human B-cell translocation gene 2, regulates expansions of stage-specific thymocytes and hematopoietic progenitors. In the present study, lineage-negative (Lin(-))/stem cell antigen-1-positive (Sca-1+)/c-Kit+ (LSK) cell content was significantly elevated in bone marrow (BM) of TIS21-knockout (TIS21(-/-)) female mice, suggesting 17beta-estradiol (E(2))-regulated progenitor expansion. E(2) induced DNA synthesis and cell proliferation of mouse embryonic fibroblasts (MEFs) isolated from TIS21(-/-) mice, but not wild type (WT). In contrast to WT, E(2) failed to activate protein kinase B (Akt) in the TIS21(-/-) MEFs, independent of extracellular signal-regulated kinase 1/2 (Erk1/2) activation. Despite attenuation of Akt activation, mammalian target of rapamycin (mTOR) was constitutively activated in the TIS21(-/-) MEFs. Furthermore, mitogen-activated protein kinase 1/2 inhibitor or knockdown of Erk1 could restore activation of Akt and downregulate mTOR. Immunoprecipitation showed Akt preferentially bound to phosphorylated Erk1/2 (p-Erk1/2) in TIS21(-/-) cells, but reconstitution of TIS21 inhibited their interaction. E(2)-injected TIS21(-/-) male mice also increased LSK cells in BM. Taken together, expansion of hematopoietic progenitors in TIS21(-/-) female mice might be through inhibition of Akt activation, and constitutive activation of mTOR via preferential binding of TIS21 to E(2)-induced p-Erk1/2, compared with that of Akt. Our results suggest that TIS21 plays a pivotal role in maintaining the hematopoietic stem cell compartment and hematopoiesis.     

Circulating hematopoietic progenitors with T lineage potential

The thymus is seeded via the blood, but the identity of hematopoietic progenitors with access to the circulation in adult mice is unknown. We report here that the only progenitors in blood with efficient T lineage potential were lineage negative with high expression of stem cell antigen 1 and c-Kit (LSK cells). The blood LSK population, like its counterpart in the bone marrow, contained hematopoietic stem cells and nonrenewing, multipotent progenitors, including early lymphoid progenitors and CD62L(+) cells previously described as efficient T lineage progenitors. Common lymphoid progenitors could not be identified in the circulation, suggesting they are not physiological T lineage precursors. We conclude that blood LSK cells are the principal circulating progenitors with T lineage potential.     

Jagged2-expressing hematopoietic progenitors promote regulatory T cell expansion in the periphery through notch signaling

Cellular interactions promoting the in vivo expansion of CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells for maintenance of immune tolerance remain poorly defined. Here we report that mobilized Lin(-)Sca-1(+)c-kit(+) (LSK) hematopoietic progenitor cells (HPCs), unlike medullary hematopoietic stem cells (HSCs), selectively drove the direct, immediate expansion of functional host-derived Treg cells, thereby preventing the progression to overt spontaneous autoimmune diabetes in nonobese diabetic mice. Treg cell expansion required cell-to-cell contact and Notch3 signaling, which was mediated selectively through the Notch ligand Jagged2 expressed by the multipotent HPC subset, as assessed by small interfering RNA (siRNA) silencing. Conversely, notwithstanding their similar multilineage microchimerism, neither sorted Jagged2(-) HPCs nor Jagged2(lo) medullary HSCs were able to expand Treg cells. These data provide evidence for a productive Notch-mediated interaction between a unique subset of mobilized hematopoietic progenitors and Treg cells. They open therapeutic perspectives for autologous transplantation of Jagged2(+) LSK progenitors to promote Treg cell expansion in T cell-mediated diseases.     

miR‐221 redirects precursor B cells to the BM and regulates their residence

Pluripotent hematopoietic stem cells and multipotent myeloid/lymphoid progenitors express miR‐221 and miR‐222. When Pax5 expression commits these progenitors to monopotent pre‐B lymphocytes the two microRNAs (miRNAs) are downregulated. Upon transplantation, stem cells and progenitors can reside in the BM, while pre‐B cells, after their commitment, no longer do so. Retrovirally transduced, doxycycline‐induced overexpression of either miR‐221 or miR‐222 in pre‐B‐I cells does not revert their monopotency to multipotency. However, upon transplantation miR‐221, but not miR‐222, transduced pre‐B‐I cells regain the capacity to reside in the BM. Upon subsequent termination of miR‐221‐expression by removal of doxycycline, the transplanted cells leave the BM again. Microarray analyses identified 25 downregulated miR‐221‐target genes, which could function to localize phases of B‐lymphocyte development in BM before and after commitment.     

Endoglin is not critical for hematopoietic stem cell engraftment and reconstitution but regulates adult erythroid development

Endoglin is a transforming growth factor-beta (TGF-beta) accessory receptor recently identified as being highly expressed on long-term repopulating hematopoietic stem cells (HSC). However, little is known regarding its function in these cells. We have used two complementary approaches toward understanding endoglin's role in HSC biology: one that efficiently knocks down expression via lentiviral-driven short hairpin RNA and another that uses retroviral-mediated overexpression. Altering endoglin expression had functional consequences for hematopoietic progenitors in vitro such that endoglin-suppressed myeloid progenitors (colony-forming unit-granulocyte macrophage) displayed a higher degree of sensitivity to TGF-beta-mediated growth inhibition, whereas endoglin-overexpressing cells were partially resistant. However, transplantation of transduced bone marrow enriched in primitive hematopoietic stem and progenitor cells revealed that neither endoglin suppression nor endoglin overexpression affected the ability of stem cells to short-term or long-term repopulate recipient marrow. Furthermore, transplantation of cells altered in endoglin expression yielded normal white blood cell proportions and peripheral blood platelets. Interestingly, decreasing endoglin expression increased the clonogenic capacity of early blast-forming unit-erythroid progenitors, whereas overexpression compromised erythroid differentiation at the basophilic erythroblast phase, suggesting a pivotal role for endoglin at key stages of adult erythropoietic development.     

Cyclophosphamide promotes engraftment of gene-modified cells in a mouse model of Fanconi anemia without causing cytogenetic abnormalities

A major hurdle for hematopoietic stem cell (HSC) gene therapy for inherited bone marrow disorders, including Fanconi anemia (FA), is adequate engraftment of gene-modified cells. A phenotypic defect in DNA repair renders FA patients sensitive to alkylating agents such as cyclophosphamide (Cy); however, at lower doses, Cy is well tolerated in the FA transplant setting. We tested whether non-alkylating agents could replace Cy for pretransplant conditioning to enhance engraftment of FANCA gene-modified hematopoietic cells. We compared Cy preconditioning with fludarabine (Flu) or cytarabine (AraC) or no conditioning as a control in fanca ^?/? mutant mice receiving gene-modified bone marrow cells. Only mice conditioned with Cy exhibited appreciable engraftment of gene-modified cells by PCR and resistance to mitomycin C (MMC). Cy administration following transplantation increased gene marking levels in all animals treated, but highest gene marking and corresponding MMC resistance were observed in mice receiving Cy pre- and posttransplantation. Importantly, no cytogenetic abnormalities were observed in Cy-treated mice. We conclude that Cy is an effective and superior preparative regimen with respect to engraftment of lentivirus-transduced cells and functional correction in fanca ^?/? mice. Thus, appropriately dosed Cy may provide a suitable conditioning regimen for FA patients undergoing HSC gene therapy.     

Failure of transdifferentiation of adult hematopoietic stem cells into neurons

Previous studies of bone marrow-derived stem cell transdifferentiation into neurons have not involved purified cell populations and determined their exact phenotype prior to differentiation. The present study investigates whether highly purified mouse adult hematopoietic stem cells (HSCs), characterized by lineage marker depletion and expression of the cell surface markers Sca1 and c-Kit (Lin(-) Sca1(+) c-Kit(+) [LSK]), can be stimulated to adopt a neuronal fate. When the HSC(LSK) cells were cultured in vitro in neuronal differentiation medium supplemented with retinoic acid, 50% of the cells expressed the neural progenitor marker nestin and no cells had become postmitotic. Electrophysiological recordings on neuron-like cells showed that these cells were incapable of generating action potentials. When the HSC(LSK) cells either were grown in vitro together with neural precursor cells or were transplanted into the striatum or cerebellum of wild-type mouse, they either differentiated into Iba1-immunopositive macrophage/microglia or died. In conclusion, we demonstrate that adult HSC(LSK) cells do not have the capacity to leave the hematopoietic lineage and differentiate into neurons.     

Lack of correlation between an assay used to determine early marrow allograft rejection and long-term chimerism after murine allogeneic bone marrow transplantation: effects of marrow dose.

The acute rejection of bone marrow (BM) allografts by host effectors can occur within a short period after BM transplantation (BMT) in lethally irradiated mice. Common assays used to ascertain engraftment/resistance involve measuring the growth of granulocyte/monocyte progenitors (colony-forming unit-granulocyte-macrophage) in vitro or splenocyte proliferation assessed by radioisotope incorporation in vivo 5 to 8 days after BMT. However, the correlation of the long-term outcome of BMT with the kinetics of recovery by using the dose of allogeneic BM cells (BMCs) that leads to early rejection as determined by the in vitro assessment has not been extensively studied. Thus, to investigate whether the early rejection of donor BMCs is an indication of a long-term engraftment failure, C57BL/6 (H2b) mice were lethally irradiated and transplanted with various doses of BALB/c (H2d) BMCs. The short-term engraftment of donor precursors (colony-forming unit-granulocyte-macrophage), the kinetics of hematopoietic cell recovery, the extent of donor chimerism, and the proportion of the recipients with long-term survival were determined. The results show that the kinetics and extent of hematopoietic cell recovery were significantly delayed in mice receiving limiting doses of BMCs that were rejected or severely resisted at day 8 after BMT. However, a proportion of these mice survived up to 98 days after BMT with mixed chimerism or donor chimerism. This study demonstrates that early rejection of BM precursors, as assessed by measurement of myeloid progenitors in the spleen after BMT, does not always correlate with the long-term outcome of the marrow allograft and that significant variability is inherent in the extent of chimerism when threshold amounts of BMCs are used.     

Cytokines regulate postnatal hematopoietic stem cell expansion: opposing roles of thrombopoietin and LNK

The role of cytokines as regulators of hematopoietic stem cell (HSC) expansion remains elusive. Herein, we identify thrombopoietin (THPO) and the cytokine signaling inhibitor LNK, as opposing physiological regulators of HSC expansion. Lnk(-/-) HSCs continue to expand postnatally, up to 24-fold above normal by 6 mo of age. Within the stem cell compartment, this expansion is highly selective for self-renewing long-term HSCs (LT-HSCs), which show enhanced THPO responsiveness. Lnk(-/-) HSC expansion is dependent on THPO, and 12-wk-old Lnk(-/-)Thpo(-/-) mice have 65-fold fewer LT-HSCs than Lnk(-/-) mice. Expansions of multiple myeloid, but not lymphoid, progenitors in Lnk(-/-) mice also proved THPO-dependent.     

Resolving Salmonella infection reveals dynamic and persisting changes in murine bone marrow progenitor cell phenotype and function

The generation of immune cells from BM precursors is a carefully regulated process. This is essential to limit the potential for oncogenesis and autoimmunity yet protect against infection. How infection modulates this is unclear. Salmonella can colonize systemic sites including the BM and spleen. This resolving infection has multiple IFN‐γ‐mediated acute and chronic effects on BM progenitors, and during the first week of infection IFN‐γ is produced by myeloid, NK, NKT, CD4⁺ T cells, and some lineage‐negative cells. After infection, the phenotype of BM progenitors rapidly but reversibly alters, with a peak ～30‐fold increase in Sca‐1^hi progenitors and a corresponding loss of Sca‐1^lo/int subsets. Most strikingly, the capacity of donor Sca‐1^hi cells to reconstitute an irradiated host is reduced; the longer donor mice are exposed to infection, and Sca‐1^hic‐kit^int cells have an increased potential to generate B1a‐like cells. Thus, Salmonella can have a prolonged influence on BM progenitor functionality not directly related to bacterial persistence. These results reflect changes observed in leucopoiesis during aging and suggest that BM functionality can be modulated by life‐long, periodic exposure to infection. Better understanding of this process could offer novel therapeutic opportunities to modulate BM functionality and promote healthy aging.     

The role of the chromatin remodeler Mi-2beta in hematopoietic stem cell self-renewal and multilineage differentiation

The ability of somatic stem cells to self-renew and differentiate into downstream lineages is dependent on specialized chromatin environments that keep stem cell-specific genes active and key differentiation factors repressed but poised for activation. The epigenetic factors that provide this type of regulation remain ill-defined. Here we provide the first evidence that the SNF2-like ATPase Mi-2beta of the Nucleosome Remodeling Deacetylase (NuRD) complex is required for maintenance of and multilineage differentiation in the early hematopoietic hierarchy. Shortly after conditional inactivation of Mi-2beta, there is an increase in cycling and a decrease in quiescence in an HSC (hematopoietic stem cell)-enriched bone marrow population. These cycling mutant cells readily differentiate into the erythroid lineage but not into the myeloid and lymphoid lineages. Together, these effects result in an initial expansion of mutant HSC and erythroid progenitors that are later depleted as more differentiated proerythroblasts accumulate at hematopoietic sites exhibiting features of erythroid leukemia. Examination of gene expression in the mutant HSC reveals changes in the expression of genes associated with self-renewal and lineage priming and a pivotal role of Mi-2beta in their regulation. Thus, Mi-2beta provides the hematopoietic system with immune cell capabilities as well as with an extensive regenerative capacity.     

Bone Marrow Mesenchymal Stem Cells Carrying FANCD2 Mutation Differ from the Other Fanconi Anemia Complementation Groups in Terms of TGF-β1 Production

Transforming growth factor beta (TGF-β) secretion from cells in the bone marrow (BM) niche affects hematopoietic stem cell (HSC) fate and has a cardinal role in HSC quiescence. BM mesenchymal stem cells (BM-MSCs), a component of the BM niche, may produce abnormal levels of TGF-β in Fanconi anemia (FA) and may play a role in bone marrow failure. Here, we molecularly and cellularly characterized FA BM-MSCs by addressing their immunophenotype, proliferation- and differentiation- capacity, reactive oxygen species (ROS) production, senescence activity as well as expression and secretion levels of TGF-β isoforms. In ten FA patients, mutations were detected in FANCA (n?=?7), FANCG (n?=?1) and FANCD2 (n?=?2) genes. The immunophenotype, with the exception of CD29, and differentiation capacity of FA BM-MSCs were similar to healthy donors. FA BM-MSCs showed decreased proliferation, increased ROS level and an arrest in G2 following DEB treatment. β-galactosidase staining indicated elevated senescence of FANCD2-deficient cells. FA BM-MSCs displayed TGF-β1 mRNA levels similar to donor BM-MSCs, and was not affected by DEB treatment. However, secretion of TGF-β was absent in FA-D2 BM-MSCs. Absence of TGF-β secretion may be related to early onset of senescence of the FANCD2-deficient BM-MSCs. The proliferative response of FA-D2 BM-MSCs to rTGF-β1 was not different from FANCA-deficient and donor cells and raises the possibility that rTGF-β1 may reverse the senescence of the FANCD2-deficient BM-MSCs which needs to be investigated further.