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.

Renal and Hematological Effects of CLCF-1, a B-Cell-Stimulating Cytokine of the IL-6 Family.

CLCF-1 is a cytokine known for B-cell stimulation and for neurotrophic properties. We have identified CLCF-1 as a potential injurious factor in the human renal disease focal segmental glomerulosclerosis (FSGS). We investigated its effects on renal cells and renal function in in vitro and in vivo studies. Methods include measurement of the effect of CLCF-1 on phosphorylation of target molecules of the JAK/STAT pathway, on cytoskeleton and cell morphology in cultured podocytes, on albumin permeability of isolated rat glomeruli, and on tissue phosphorylation and urine albumin after acute or chronic CLCF-1 injection. In addition, cell sorting was performed to determine the presence of cells expressing CLCF-1 in spleen and bone marrow of normal mice and the effect of CLCF-1 infusion on splenic B-cell populations. CLCF-1 increased phosphorylation of STAT3 in multiple cell types, activated podocytes leading to formation of lamellipodia and decrease in basal stress fibers, increased glomerular albumin permeability, and increased STAT3 phosphorylation of peripheral blood cells and renal cortex. CLCF-1 increased urine albumin/creatinine ratio in mice and increased B-cell expression of IgG in mouse spleen. We conclude that CLCF-1 has potentially important systemic effects, alters podocyte function, and may contribute to renal dysfunction and albuminuria.

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.

Role of mesenchymal stem cell therapy in Crohn's disease.

Many trials of mesenchymal stem cells (MSCs) have been published in the past 5-6 y. MSCs inhibit T-cell alloreactivity in vitro by soluble factors and direct cell-to-cell contact. They are safe to infuse in humans with no acute toxicity and no ectopic tissue formation. Promising results of MSC infusion for graft-vs.-host disease and fistulizing Crohn's disease (CD) have been published. Treatment of CD requires a comprehensive treatment approach to maintain symptomatic control, improve health-related quality-of-life measures, and minimize complications from the disease. In this review, we will discuss the results of clinical trials using a novel treatment in the form of MSCs for treatment of CD and related complications. Success of these phase I, II, and III trials have set the stage for usage of this novel treatment for children with CD.

Emerging uses for pediatric hematopoietic stem cells.

Many new therapies are emerging that use hematopoietic stem and progenitor cells. In this review, we focus on five promising emerging trends that are altering stem cell usage in pediatrics: (i) The use of hematopoietic stem cell (HSC) transplantation, autologous or allogeneic, in the treatment of autoimmune disorders is one. (ii) The use of cord blood transplantation in patients with inherited metabolic disorders such as Hurler syndrome shows great benefit, even more so than replacement enzyme therapy. (iii) Experience with the delivery of gene therapy through stem cells is increasing, redefining the potential and limitations of this therapy. (iv) It has recently been shown that human immunodeficiency virus (HIV) infection can be cured by the use of selected stem cells. (v) Finally, it has long been postulated that HSC-transplantation can be used to induce tolerance in solid-organ transplant recipients. A new approach to tolerance induction using myeloid progenitor cells will be described.

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.

Renal failure causes early death of bcl-2 deficient mice.

BCL-2 functions as a death repressor molecule in an evolutionary conserved cell death pathway. Inactivation of bcl-2 in mice results in pleiotropic effects including postnatal growth retardation, massive apoptosis in lymphoid tissues, polycystic kidney disease (PKD) and shortened lifespan. To evaluate the influence of the affected bcl-2 deficient kidneys on the postnatal development and lifespan of bcl-2 knockout mice we used "the rescue of (n-1) affected tissues" strategy. According to this strategy bcl-2 heterozygous animals were crossed with H2K-hbcl-2 transgenic mice expressing human BCL-2 in most tissues and organs excluding the kidney. Overexpression of hBCL-2 in bcl-2-/- mice rescues growth retardation, normalizes and protects the hematolymphoid system from gamma-radiation. However, the hbcl-2 transgene is not expressed in kidneys and the rescued mice have PKD and a shortened lifespan. Thus, our results indicated that PKD is the main reason of early mortality in bcl-2 deficient mice. Moreover, we have created mouse model, similar to the kidney specific knockout of bcl-2. Such models can be useful to study the influence of bcl-2 or other gene deficiency in individual organs (or tissues) on development and ageing of whole organism.

Costimulation with interleukin-4 and interleukin-10 induces mast cell apoptosis and cell-cycle arrest: the role of p53 and the mitochondrion.

OBJECTIVE: The aim of this study was to determine the mechanism by which interleukin (IL)-4 + IL-10 costimulation regulates mast cell numbers to maintain immune homeostasis. MATERIALS AND METHODS: We employed mouse bone marrow-derived mast cells (BMMC) to measure the effects of IL-4 + IL-10 on survival and cell-cycle progression. p53-Deficient, bax-deficient, and bcl-2 transgenic BMMC were compared to wild-type cells to determine the role of these proteins in apoptosis. The molecular regulation of apoptosis and cell-cycle progression was investigated using flow cytometric analysis, RNase protection, and Western blotting. RESULTS: IL-4 + IL-10 induced BMMC apoptosis and arrest. Apoptosis was p53-dependent. Cell death was accompanied by loss of mitochondrial membrane potential, the importance of which was demonstrated by resistance to IL-4 + IL-10-mediated cell death when Bax was deleted or Bcl-2 was overexpressed. Those cells not killed by apoptosis demonstrated a p53-independent G1 cell-cycle arrest. Apoptosis and arrest may be explained by reduced IL-3 receptor signaling. CONCLUSION: Costimulation with IL-4 + IL-10 partly controls mast cell homeostasis through a delayed apoptosis and arrest program that is induced by a blockade of IL-3 receptor signaling. The delay in these negative effects would allow the protective effects of mast cell activation to occur for several days.

Hematopoietic stem cell dose correlates with the speed of immune reconstitution after stem cell transplantation.

In the current study, we tested whether higher numbers of hematopoietic stem cells correlate with the speed of immune reconstitution in a congenic transplantation model (C57BL/Ka, CD45.1, Thy1.1-->C57BL/6, CD45.2, Thy1.2) using purified hematopoietic stem cells (c-Kit(+)Thy1.1(low)Lin(-/low)Sca-1(+)). There were 3 different doses of stem cells used (400, 1000, and 5000). Phenotypic analyses in peripheral blood and spleen demonstrated that higher numbers of infused stem cells are associated with more rapid regeneration of T cells (CD4(+), CD8(+), naive CD4(+), naive CD8(+)) and B cells at early time points. The numbers of T and B cells eventually became equivalent between different dose groups at late time points. Production of interleukin-2 and inter-feron-gamma per T cell was similar regardless of stem cell dose even when tested at the time when there were significant differences in peripheral T-cell counts. The improved immune recovery was attributed to a more rapid regeneration of donor-type immune cells. Higher numbers of total thymocytes and signal joint T-cell receptor excision circles were observed in the higher dose stem cell recipients, suggesting that accelerated regeneration of T cells was due to enhanced thymopoiesis.

Interleukin-4 elicits apoptosis of developing mast cells via a Stat6-dependent mitochondrial pathway.

OBJECTIVE: The aim of this study was to assess the effects of interleukin-4 and signal transducer and activator of transcription (Stat)-6 on IL-3+SCF-induced mast cell development. PATIENTS AND METHODS: Unseparated mouse bone marrow cells were cultured in IL-3+SCF, giving rise to mast cells and monocytes/macrophages. The addition of IL-4, the use of Stat6-deficient bone marrow cells, and expression of a constitutively active Stat6 mutant were employed to assess the effects of IL-4 and Stat6 on cell viability, proliferation, and differentiation. Bax-deficient and bcl-2 transgenic bone marrow cells were used to assess the importance of the mitochondria in IL-4-mediated effects. RESULTS: IL-4 elicited apoptosis and limited the cell cycle progression of developing bone marrow cells, without affecting cell differentiation. Apoptosis required that IL-4 be present during the first 8 days of the 21-day culture period. Cell death correlated with loss of mitochondrial membrane potential. Accordingly, IL-4-mediated apoptosis was inhibited by Bax deletion or bcl-2 overexpression. Lastly, Stat6 activation was both necessary and sufficient to inhibit cell survival. CONCLUSION: IL-4 exerts potent apoptotic effects on developing mast cells and monocyte/macrophages through mitochondrial damage and Stat6 activation.

Hematopoietic stem cells and other hematopoietic cells show broad resistance to chemotherapeutic agents in vivo when overexpressing bcl-2.

Objective. Chemotherapeutic agents function by inducing apoptosis and their effectiveness depends on the balance of pro- and anti-apoptotic proteins in cells. Due to the complicated interactions of the many proteins involved, it has been difficult to determine in tumors whether overexpression of single genes is prognostic for increased resistance. Therefore, we studied the influence of bcl-2 overexpression on resistance to chemotherapeutics in a transgenic mouse system. This allowed us to study a wide variety of cells, including important but rare populations such as hematopoietic stem cells (HSC).Methods. H2K-bcl-2 transgenic and wild-type (WT) mice were treated with several agents(5-fluoruracil, cyclophosphamide, and busulfan) to determine the contribution of increased amounts of bcl-2 to the response to these chemotherapeutics in vivo. Populations were enumerated using flow cytometry. HSC were studied by FACS purification and long-term reconstitution assays in vivo and resistance was confirmed by short-term proliferation assays with different amounts of chemotherapeutics in vitro.Results. bcl-2 overexpression alone protects many cell types, though protection levels differ between populations and agents. However, even sensitive populations return to pretreatment levels faster in transgenic mice. bcl-2 overexpression also prevents the dramatic changes in HSC following 5-FU treatment (downregulation of c-kit, upregulation of Lin, less efficient long-term reconstitution). In vitro studies directly demonstrate increased resistance of bcl-2 overexpressing HSC to chemotherapeutic agents.Conclusions. Increased expression of bcl-2 in HSC and their progeny endows these cells with broad resistance to chemotherapeutic agents. The ability to (differentially) regulate sensitivity to apoptosis of bystander and tumor cells is clinically important.

A role for Wnt signalling in self-renewal of haematopoietic stem cells.

Haematopoietic stem cells (HSCs) have the ability to renew themselves and to give rise to all lineages of the blood; however, the signals that regulate HSC self-renewal remain unclear. Here we show that the Wnt signalling pathway has an important role in this process. Overexpression of activated beta-catenin expands the pool of HSCs in long-term cultures by both phenotype and function. Furthermore, HSCs in their normal microenvironment activate a LEF-1/TCF reporter, which indicates that HCSs respond to Wnt signalling in vivo. To demonstrate the physiological significance of this pathway for HSC proliferation we show that the ectopic expression of axin or a frizzled ligand-binding domain, inhibitors of the Wnt signalling pathway, leads to inhibition of HSC growth in vitro and reduced reconstitution in vivo. Furthermore, activation of Wnt signalling in HSCs induces increased expression of HoxB4 and Notch1, genes previously implicated in self-renewal of HSCs. We conclude that the Wnt signalling pathway is critical for normal HSC homeostasis in vitro and in vivo, and provide insight into a potential molecular hierarchy of regulation of HSC development.

Enforced expression of Bcl-2 restores the number of NK cells, but does not rescue the impaired development of NKT cells or intraepithelial lymphocytes, in IL-2/IL-15 receptor beta-chain-deficient mice.

IL-2/IL-15Rbeta-deficient mice display impaired development of NK cells, NKT cells, and intraepithelial lymphocytes of the intestine and skin. To determine the role of survival signals mediated by IL-2/IL-15R in the development of these innate lymphocytes, we introduced a bcl-2 transgene into IL-2/IL-15Rbeta-deficient mice. Enforced expression of Bcl-2 restored the number of NK cells in IL-2/IL-15Rbeta-deficient mice, but the rescued NK cells showed no cytotoxic activity. The numbers of NKT cells and intestinal intraepithelial lymphocytes did not increase significantly, and skin intraepithelial lymphocytes remained undetectable in the bcl-2 transgenic IL-2/IL-15Rbeta-deficient mice. These results indicate an essential role of IL-2/IL-15R-mediated survival signals in the development of NK cells, but they also show that additional nonsurvival signals from IL-2/IL-15R are necessary for innate lymphocyte development.