Open-Label Phase II Prospective, Randomized, Controlled Study of Romyelocel-L Myeloid Progenitor Cells to Reduce Infection During Induction Chemotherapy for Acute Myeloid Leukemia.

PURPOSE: Standard cytotoxic induction chemotherapy for acute myeloid leukemia (AML) results in prolonged neutropenia and risk of infection. Romyelocel-L is a universal, allogeneic myeloid progenitor cell product being studied to reduce infection during induction chemotherapy. PATIENTS AND METHODS: One hundred sixty-three patients with de novo AML (age ≥ 55 years) receiving induction chemotherapy were randomly assigned on day 0 (d0), of whom 120 were evaluable. Subjects received either romyelocel-L infusion on d9 with granulocyte colony-stimulating factor (G-CSF) starting daily d14 (treatment group) or G-CSF daily alone on d14 (control) until absolute neutrophil count recovery to 500/µL. End points included days in febrile episode, microbiologically defined infections, clinically diagnosed infection, and days in hospital. RESULTS: Mean days in febrile episode was shorter in the treatment arm from d15 through d28 (2.36 v 3.90; P = .02). Similarly, a trend toward decreased microbiologically defined infections and clinically diagnosed infection in the treatment arm was observed from d9 to d28 (35.6% v 47.5%; P = .09), reaching a statistically significant difference from d15 to d28 (6.8% v 27.9%; P = .002). Because of this, antibacterial or antifungal use for treatment of an infection was significantly less in the treatment group (d9-d28: 44.1% v 63.9%; P = .01). Significantly fewer patients in the treatment arm received empiric antifungals from d9 tod28 (42.4% v 63.9%; P = .02) and d15-d28 (42.4% v 62.3%; P = .02). Patients in the treatment arm also had 3.2 fewer hospital days compared with control (25.5 v 28.7; P = .001). Remission rates and days to absolute neutrophil count recovery were similar in the two groups. No patients in the romyelocel-L plus G-CSF group died because of infection compared with two patients in the control arm. No graft-versus-host disease was observed. CONCLUSION: Subjects receiving romyelocel-L showed a decreased incidence of infections, antimicrobial use, and hospitalization, suggesting that romyelocel-L may provide a new option to reduce infections in patients with AML undergoing induction therapy.

Sequentially inducible mouse models reveal that Npm1 mutation causes malignant transformation of Dnmt3a-mutant clonal hematopoiesis.

Clonal hematopoiesis (CH) is a common aging-associated condition with increased risk of hematologic malignancy. Knowledge of the mechanisms driving evolution from CH to overt malignancy has been hampered by a lack of in vivo models that orthogonally activate mutant alleles. Here, we develop independently regulatable mutations in DNA methyltransferase 3A (Dnmt3a) and nucleophosmin 1 (Npm1), observed in human CH and AML, respectively. We find Dnmt3a mutation expands hematopoietic stem and multipotent progenitor cells (HSC/MPPs), modeling CH. Induction of mutant Npm1 after development of Dnmt3a-mutant CH causes progression to myeloproliferative disorder (MPD), and more aggressive MPD is observed with longer latency between mutations. MPDs uniformly progress to acute myeloid leukemia (AML) following transplant, accompanied by a decrease in HSC/MPPs and an increase in myeloid-restricted progenitors, the latter of which propagate AML in tertiary recipient mice. At a molecular level, progression of CH to MPD is accompanied by selection for mutations activating Ras/Raf/MAPK signaling. Progression to AML is characterized by additional oncogenic signaling mutations (Ptpn11, Pik3r1, Flt3) and/or mutations in epigenetic regulators (Hdac1, Idh1, Arid1a). Together, our study demonstrates that Npm1 mutation drives evolution of Dnmt3a-mutant CH to AML and rate of disease progression is accelerated with longer latency of CH.

Bone Marrow-Derived Proangiogenic Cells Mediate Pulmonary Arteriole Stiffening via Serotonin 2B Receptor Dependent Mechanism.

RATIONALE: Pulmonary arterial hypertension is a deadly disease of the pulmonary vasculature for which no disease-modifying therapies exist. Small-vessel stiffening and remodeling are fundamental pathological features of pulmonary arterial hypertension that occur early and drive further endovascular cell dysfunction. Bone marrow (BM)-derived proangiogenic cells (PACs), a specialized heterogeneous subpopulation of myeloid lineage cells, are thought to play an important role in pathogenesis. OBJECTIVE: To determine whether BM-derived PACs directly contributed to experimental pulmonary hypertension (PH) by promoting small-vessel stiffening through 5-HT2B (serotonin 2B receptor)-mediated signaling. METHODS AND RESULTS: We performed BM transplants using transgenic donor animals expressing diphtheria toxin secondary to activation of an endothelial-specific tamoxifen-inducible Cre and induced experimental PH using hypoxia with SU5416 to enhance endovascular injury and ablated BM-derived PACs, after which we measured right ventricular systolic pressures in a closed-chest procedure. BM-derived PAC lineage tracing was accomplished by transplanting BM from transgenic donor animals with fluorescently labeled hematopoietic cells and treating mice with a 5-HT2B antagonist. BM-derived PAC ablation both prevented and reversed experimental PH with SU5416-enhanced endovascular injury, reducing the number of muscularized pulmonary arterioles and normalizing arteriole stiffness as measured by atomic force microscopy. Similarly, treatment with a pharmacological antagonist of 5-HT2B also prevented experimental PH, reducing the number and stiffness of muscularized pulmonary arterioles. PACs accelerated pulmonary microvascular endothelial cell injury response in vitro, and the presence of BM-derived PACs significantly correlated with stiffer pulmonary arterioles in pulmonary arterial hypertension patients and mice with experimental PH. RNA sequencing of BM-derived PACs showed that 5-HT2B antagonism significantly altered biologic pathways regulating cell proliferation, locomotion and migration, and cytokine production and response to cytokine stimulus. CONCLUSIONS: Together, our findings illustrate that BM-derived PACs directly contribute to experimental PH with SU5416-enhanced endovascular injury by mediating small-vessel stiffening and remodeling in a 5-HT2B signaling-dependent manner.

Expansion of Umbilical Cord Blood Aldehyde Dehydrogenase Expressing Cells Generates Myeloid Progenitor Cells that Stimulate Limb Revascularization.

Uncompromised by chronic disease-related comorbidities, human umbilical cord blood (UCB) progenitor cells with high aldehyde dehydrogenase activity (ALDHhi cells) stimulate blood vessel regeneration after intra-muscular transplantation. However, implementation of cellular therapies using UCB ALDHhi cells for critical limb ischemia, the most severe form of severe peripheral artery disease, is limited by the rarity (<0.5%) of these cells. Our goal was to generate a clinically-translatable, allogeneic cell population for vessel regenerative therapies, via ex vivo expansion of UCB ALDHhi cells without loss of pro-angiogenic potency. Purified UCB ALDHhi cells were expanded >18-fold over 6-days under serum-free conditions. Consistent with the concept that ALDH-activity is decreased as progenitor cells differentiate, only 15.1% ± 1.3% of progeny maintained high ALDH-activity after culture. However, compared to fresh UCB cells, expansion increased the total number of ALDHhi cells (2.7-fold), CD34+ /CD133+ cells (2.8-fold), and hematopoietic colony forming cells (7.7-fold). Remarkably, injection of expanded progeny accelerated recovery of perfusion and improved limb usage in immunodeficient mice with femoral artery ligation-induced limb ischemia. At 7 or 28 days post-transplantation, mice transplanted with expanded ALDHhi cells showed augmented endothelial cell proliferation and increased capillary density compared to controls. Expanded cells maintained pro-angiogenic mRNA expression and secreted angiogenesis-associated growth factors, chemokines, and matrix modifying proteins. Coculture with expanded cells augmented human microvascular endothelial cell survival and tubule formation under serum-starved, growth factor-reduced conditions. Expanded UCB-derived ALDHhi cells represent an alternative to autologous bone marrow as an accessible source of pro-angiogenic hematopoietic progenitor cells for the refinement of vascular regeneration-inductive therapies. Stem Cells Translational Medicine 2017;6:1607-1619.

Cryopreserved Ex Vivo-Expanded Allogeneic Myeloid Progenitor Cell Product Protects Neutropenic Mice From a Lethal Fungal Infection.

Severe neutropenia induced by chemotherapy or conditioning for hematopoietic cell transplantation often results in morbidity and mortality due to infection by opportunistic pathogens. A system has been developed to generate ex vivo-expanded mouse myeloid progenitor cells (mMPCs) that produce functional neutrophils in vivo upon transplantation in a pathogen challenge model. It has previously been demonstrated that transplantation of large numbers of freshly isolated myeloid progenitors from a single donor provides survival benefit in radiation-induced neutropenic mice. In the present work, an ex vivo-expanded and cryopreserved mMPC product generated from an allogeneic donor pool retains protective activity in vivo in a lethal fungal infection model. Infusion of the allogeneic pooled mMPC product is effective in preventing death from invasive Aspergillus fumigatus in neutropenic animals, and protection is dose dependent. Cell progeny from the mMPC product is detected in the bone marrow, spleen, blood, and liver by flow cytometry 1 week postinfusion but is no longer evident in most animals 4 weeks posttransplant. In this model, the ex vivo-generated pooled allogeneic mMPC product (i) expands and differentiates in vivo; (ii) is functional and prevents death from invasive fungal infection; and (iii) does not permanently engraft or cause allosensitization. These data suggest that an analogous ex vivo-expanded human myeloid progenitor cell product may be an effective off-the-shelf bridging therapy for the infectious complications that develop during hematopoietic recovery following hematopoietic cell transplantation or intensive chemotherapy.

Rapid tolerance induction by hematopoietic progenitor cells in the absence of donor-matched lymphoid cells.

BACKGROUND: Donor specific hematopoietic cell transplantation has long been recognized for its potential in tolerance induction for subsequently transplanted organs. We have recently published that co-administration of Myeloid Progenitor (MP) and third party Hematopoietic Stem Cells (HSC) can induce MP-specific tolerance for subsequently transplanted organs [1]. METHODS: Mice received an allogeneic HSC and third party MP transplantation simultaneous with placement of a MP-matched skin graft. Variants tested include time of graft placement, MP genotype and source of cells. RESULTS: Using B10;B6-Rag2(-/-)Il2rg(-/-) mice, we demonstrate that specific tolerance can be induced by MP given simultaneous with the skin graft in the complete absence of MP-donor-matched lymphoid cells. Ex vivo expanded MP function as well as sorted cells in inducing tolerance. In addition we demonstrate that tolerance can be induced by MP in the context of autologous HSC transplantation. CONCLUSIONS: Our results demonstrate that the previously observed expansion of organ donor matched Treg is not essential for tolerance, and that MP tolerance protocols can be envisioned in most clinical settings, including those involving deceased donor organs.

Heme oxygenase-1 derived carbon monoxide permits maturation of myeloid cells.

Critical functions of the immune system are maintained by the ability of myeloid progenitors to differentiate and mature into macrophages. We hypothesized that the cytoprotective gas molecule carbon monoxide (CO), generated endogenously by heme oxygenases (HO), promotes differentiation of progenitors into functional macrophages. Deletion of HO-1, specifically in the myeloid lineage (Lyz-Cre:Hmox1(flfl)), attenuated the ability of myeloid progenitors to differentiate toward macrophages and decreased the expression of macrophage markers, CD14 and macrophage colony-stimulating factor receptor (MCSFR). We showed that HO-1 and CO induced CD14 expression and efficiently increased expansion and differentiation of myeloid cells into macrophages. Further, CO sensitized myeloid cells to treatment with MCSF at low doses by increasing MCSFR expression, mediated partially through a PI3K-Akt-dependent mechanism. Exposure of mice to CO in a model of marginal bone marrow transplantation significantly improved donor myeloid cell engraftment efficiency, expansion and differentiation, which corresponded to increased serum levels of GM-CSF, IL-1α and MCP-1. Collectively, we conclude that HO-1 and CO in part are critical for myeloid cell differentiation. CO may prove to be a novel therapeutic agent to improve functional recovery of bone marrow cells in patients undergoing irradiation, chemotherapy and/or bone marrow transplantation.

Passive transfer of tumour-derived MDSCs inhibits asthma-related airway inflammation.

Myeloid-derived suppressor cells (MDSCs), a heterogeneous population including myeloid progenitor and immature myeloid cells, are known to inhibit T cell responses. The issue of whether tumour-derived MDSCs regulate the immune response in an asthma environment is currently unclear. Here, we have reported that tumour-derived MDSCs shift the balance back to normal in a Th2-dominant asthmatic environment. In an ovalbumin (OVA)-induced mouse asthma model, injected tumour-derived MDSCs were recruited to the lungs of asthmatic mice by CC chemokine ligand 2 (CCL2). MDSCs transferred into asthmatic mice via i.v. injection suppressed the infiltration of inflammatory cells into the lung, the Th2 cytokine, IL-4, concentration in bronchial lavage fluid and the serum level of OVA-specific IgE. Increased TGF-ß1 production in the lung was detected after transfer of MDSCs. The inhibitory effects of MDSCs were reversed upon treatment with an anti-TGF-ß1 antibody, suggesting dependence of these activities on TGF-ß1. Our findings imply that tumour-derived MDSCs inhibit the Th2 cell-mediated response against allergen in a TGF-ß1-dependent manner. Based on the collective results, we propose that asthma may be effectively targeted using a novel MDSC-based cell therapy approach.

Inflammatory arthritis increases mouse osteoclast precursors with myeloid suppressor function.

Increased osteoclastic bone resorption leads to periarticular erosions and systemic osteoporosis in RA patients. Although a great deal is known about how osteoclasts differentiate from precursors and resorb bone, the identity of an osteoclast precursor (OCP) population in vivo and its regulatory role in RA remains elusive. Here, we report the identification of a CD11b(-/lo)Ly6C(hi) BM population with OCP activity in vitro and in vivo. These cells, which can be distinguished from previously characterized precursors in the myeloid lineage, display features of both M1 and M2 monocytes and expand in inflammatory arthritis models. Surprisingly, in one mouse model of RA (adoptive transfer of SKG arthritis), cotransfer of OCP with SKG CD4+ T cells diminished inflammatory arthritis. Similar to monocytic myeloid-derived suppressor cells (M-MDSCs), OCPs suppressed CD4+ and CD8+ T cell proliferation in vitro through the production of NO. This study identifies a BM myeloid precursor population with osteoclastic and T cell-suppressive activity that is expanded in inflammatory arthritis. Therapeutic strategies that prevent the development of OCPs into mature bone-resorbing cells could simultaneously prevent bone resorption and generate an antiinflammatory milieu in the RA joint.

Labeling primitive myeloid progenitor cells in Xenopus.

In Xenopus the first blood cells to differentiate in the embryo are the primitive myeloid lineages, which arise from the anterior ventral blood islands during the neurula stages. Primitive myeloid cells (PMCs) will give rise to the embryonic pool of neutrophils and macrophages, a highly migratory population of cells with various functions during development and tissue repair. Understanding the development and behavior of PMCs depends on our ability to label, manipulate, and image these cells. Xenopus embryos have several advantages in the study of PMCs, including a well-established fate map and the possibility of performing transplants in order to label these cells. In addition, Xenopus embryos are easy to manipulate and their external development and transparency at the tadpole stages make them amenable to imaging techniques. Here we describe two methods for labeling primitive myeloid progenitor cells during early Xenopus development.

Analyzing the angiogenic potential of Gr-1(+)CD11b (+) immature myeloid cells from murine wounds.

Analysis of tissue repair and regeneration in a variety of organisms has demonstrated that stem and progenitor cells play a critical role in the healing and regenerative response. In particular, during cutaneous wound healing bone marrow-derived cells are recruited to the site of injury in large numbers, often comprising over 50% of the cells within the wound milieu. These bone marrow-derived cells are comprised mostly of a heterogeneous mix of myeloid cells. In the early stages of wound healing, the most prominent subtypes are Gr-1(+)CD11b(+) cells that consist of progenitor cells and more differentiated granulocytes. Under certain conditions, these cells have the potential to strongly promote angiogenesis, and thus tissue repair and regeneration. This chapter provides methods by which one can isolate these cells from wound tissue and assess their pro-angiogenic capacity via gene expression analyses and functional in vivo angiogenesis assays.

Myeloid derived suppressor cells in transplantation.

Myeloid derived suppressor cells (MDSC) are a heterogeneous population of hematopoietic derived cell precursors that can suppress immune responses in a variety of inflammatory settings. Here we review recent studies detailing expansion of phenotypically and functionally disparate MDSC. Findings related to MDSC accumulation, activation, and mechanisms utilized in immune suppression are presented. Further, we discuss recent reports that suggest MDSC are expanded during transplantation and that modulation of MDSC can participate in preventing graft rejection.

Tolerance induction by hematopoietic cell transplantation: combined use of stem cells and progenitor cells.

BACKGROUND: Donor-specific hematopoietic cell transplantation (HCT) in the form of bone marrow transplantation has been long recognized experimentally as a means of inducing tolerance for subsequently transplanted organs. Clinical translation has been limited, however, due to HCT-associated complications. Unrelated myeloid progenitors (MP) can be administered simultaneously with hematopoietic stem cells (HSC). This reduces susceptibility to bacterial and fungal infections in neutropenic mice in laboratory studies. It is not known, however, if the addition of third-party MP interferes with tolerance induction. METHODS: BALB/c (H-2d) mice were irradiated and reconstituted with 4,000 AKR (H-2k) HSC or with 4,000 AKR HSC combined with 10(5) FVB (H-2q) MP. After 2 months, the mice received skin grafts from these three strains or from an unrelated strain, C57BL/6 (H-2b). Composition and origin of hematopoietic cells was analyzed using flow cytometry. RESULTS: Mice in both groups accepted all the host-type- and HSC-donor-matched grafts, and rejected unrelated grafts. Surprisingly, recipients of both HSC and MP also accepted MP-matched skin grafts (14 of 14), even with very low levels of MP-derived cells in circulation. The analysis revealed that, although most hematopoietic cells were derived from HSC donors, regulatory T cells were derived from both donors as well as the recipient. CONCLUSION: The addition of third-party MP cells does not interfere with HCT-induced tolerance induction and, surprisingly, induces MP-specific tolerance.

CCR7 impairs hematopoiesis after hematopoietic stem cell transplantation increasing susceptibility to invasive aspergillosis.

Hematopoietic stem cell transplantation (HSCT) is limited by patient susceptibility to opportunistic infections. One of the most devastating infections after HSCT is invasive aspergillosis (IA), a life-threatening disease caused by Aspergillus fumigatus. Transplantation of hematopoietic stem cells (HSCs) and myeloid progenitor cells (MPCs) has been shown to mediate protection against IA, but little is known about the factors that regulate HSC and MPC cell expansion after transplantation. Herein, we investigated the role of CCR7 in a murine model of IA after combined HSC and MPC transplantation into lethally irradiated wild-type (WT) mice. Nonirradiated CCR7(-/-) mice had expanded populations of HSCs in the bone marrow and spleen, compared with WT mice. Irradiated WT mice reconstituted with CCR7(-/-) HSCs and MPCs had increased survival, decreased fungal burden, and enhanced myeloid leukocyte numbers during IA, compared with WT controls. In addition, WT mice reconstituted with WT HSCs and MPCs and treated with anti-CCR7 exhibited accelerated myeloid cell expansion similar to that observed in CCR7(-/-)→WT chimeras. Thus, removal of the inhibitory effects of CCR7 through genetic alteration or ligand immunoneutralization enhanced myeloid reconstitution, thereby accelerating fungal clearance in a murine model of IA.

Tolerogenic dendritic cells and myeloid-derived suppressor cells: potential for regulation and therapy of liver auto- and alloimmunity.

Organ transplantation is now established as an accepted treatment for end-stage liver disease, acute fulminant hepatic liver failure and hepatocellular carcinoma. While early graft acceptance rates have increased markedly due to improved immunosuppressive drug regimens, rates of late graft failure remain largely unchanged. Recent findings suggest that in addition to alloimmunity, chronic rejection of liver allografts may also reflect de novo autoimmune hepatitis or recurrence of pre-existing hepatic autoimmune disease. Dendritic cell (DC)- based therapy is a promising experimental approach to promotion of transplant tolerance and the treatment of autoimmune diseases. Newly emerging evidence also demonstrates the potential efficacy of myeloid-derived suppressor cells (MDSC) in the antigen (Ag)-specific regulation of T-cell responses. Herein, we discuss current understanding of liver autoimmunity post-transplantation, along with current approaches for the development of tolerogenic DC, and the potential use of MDSC for the development of stable, Ag-specific tolerance.

Innate immune 'self' recognition: a role for CD47-SIRPalpha interactions in hematopoietic stem cell transplantation.

Self-nonself discrimination is a central property of the immune system. This paradigm was originally established in the context of tissue transplantation, leading to the discovery of major histocompatibility complex molecules as signals of 'self'. However, accumulating evidence has shown that innate immune cells are regulated in a similar fashion. Recent evidence has suggested that interactions between the 'self' molecule CD47 and the innate inhibitory receptor signal regulatory protein-alpha expressed on macrophages may be a critical determinant of transplant engraftment, supporting the concept that 'self'-awareness is a general property of all immune cells.

[Preliminary study on immunologic tolerance for hetero-skin graft induced by chimeric donors].

OBJECTIVE: To explore the feasibility of transplanting the skin from chimeric rats to rabbits. METHODS: Chimeric rats were produced by transplanting the haematopoietic stem cells (HSCs) from rabbit marrows into fetal rats in uterus and followed by injecting the HSCs into the livers of the rats at newborn stage. After six weeks, the skin from chimeric rats was transplanted to the rabbits. In group A, the skin grafts from chimeric rat donors were transplanted to the HSCs donating rabbits, with the skin from non-chimeric rat to normal rabbits were used as control. In group B, the skin grafts from chimeric and non-chimeric rats were transplanted to the HSCs donating rabbits at the same time. Gross observation and the surviving time of heterogenic-skin graft were observed. The wound healing time was also recorded. RESULTS: In group A, the surviving time and the wound healing time of non-chimeric grafts were (9.3 +/- 1.8) days and (20.9 +/- 2.1) days, respectively, while those in chimeric grafts were (15.1 +/- 2.6) and (18.5 +/-1.3) days, respectively. In group B, the surviving time and the wound healing time of non-chimeric grafts were similar to those of group A. Compared with those in non-chimeric grafts, the surviving time of chimeric grafts in both groups were prolonged (P < 0.01), and the wound healing time shortened (P < 0.05 or 0.01). Most of the wounds healed quickly after rejection of chimeric grafts, while the wounds with non-chimeric grafts were re-opened with exudation and some necrotic tissue. CONCLUSION: Immunologic tolerance for skin graft can be induced by the skin from chimeric donors, which can prolong the surviving time of skin grafts and shorten the wound healing time.

[Experimental study of maxillary sinus lifting with tissue engineered bone].

OBJECTIVE: To evaluate the effect of tissue engineering bone in maxillary sinus lifting. METHODS: The marrow stromal stem cells of dog were cultured in DMEM containing 100 m1/L fetal bovine serum and induced to differentiate to osteoblasts, which were then inoculated together with Bio-Oss for 5 days. Sixteen dog's bilateral maxillary sinus were elevated. One side was grafted with a compound of BMSC and Bio-Oss and the other side grafted with Bio-Oss alone. The samples were studied by gross, CT, histomorphology and histomorphometrical analysis at the 30th, 90th day after the operation. t-test was used for statistical analysis. RESULTS: In gross view and CT, new bone formation was observed in all maxillary sinus after 30 and 90 days respectively. Histomorphometrical analysis showed much more new callus in BMSC-Bio-Oss group than in Bio-Oss group (P < 0.05). CONCLUSIONS: A better effect of new bone formation could be obtained with tissue engineered bone in maxillary sinus lifting.

Continuous in vivo infusion of interferon-gamma (IFN-gamma) enhances engraftment of syngeneic wild-type cells in Fanca-/- and Fancg-/- mice.

Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of FA complementation types allows the potential of using gene transfer technology to introduce functional cDNAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. However, strategies to enhance the mobilization, transduction, and engraftment of exogenous stem cells are required to optimize efficacy prior to widespread clinical use. Hypersensitivity of Fancc-/- cells to interferon-gamma (IFN-gamma), a nongenotoxic immune-regulatory cytokine, enhances engraftment of syngeneic wild-type (WT) cells in Fancc-/- mice. However, whether this phenotype is of broad relevance in other FA complementation groups is unresolved. Here we show that primitive and mature myeloid progenitors in Fanca-/- and Fancg-/- mice are hypersensitive to IFN-gamma and that in vivo infusion of IFN-gamma at clinically relevant concentrations was sufficient to allow consistent long-term engraftment of isogenic WT repopulating stem cells. Given that FANCA, FANCC, and FANCG complementation groups account for more than 90% of all FA patients, these data provide evidence that IFN-gamma conditioning may be a useful nongenotoxic strategy for myelopreparation in FA patients.

Granulocyte/macrophage origin of glomerular mesangial cells.

We previously demonstrated the ability of hematopoietic stem cells (HSCs) to generate glomerular mesangial cells by trans-planting clonal populations of cells derived from a single enhanced green fluorescent protein (EGFP)-positive HSC into lethally irradiated mice. To define more precisely the hematopoietic differentiation pathway through which mesangial cells are derived, we studied the relationship between mesangial cell expression and individual hematopoietic lineages by means of a transplantation strategy. In a series of clonal HSC transplantation experiments, we generated 3 mice engrafted predominantly by granulocytes and macrophages (GMs) and 4 mice engrafted with B-cells or with B-cells and T-cells. When the kidneys of these mice were analyzed, the mice exhibiting high GM lineage engraftment revealed much higher levels of EGFP-positive mesangial cells than those with predominantly lymphocyte engraftment. Fluorescence in situ hybridization analysis of the kidneys from a male recipient of an EGFP-positive female donor excluded cell fusion as the cause for the observed differentiation. These results support the notion that glomerular mesangial cells share their origin with GMs.