RESUMO
OBJECTIVE: The goal of this study was to determine if competitive pressure was placed on hematopoietic stem cells (HSC) by a coinjected "carrier" population that maintains short-term survival of the host. Our hypothesis was that delayed introduction of "carrier" cells would increase engraftment of donor HSC. MATERIALS AND METHODS: Competitive repopulation assays were performed using genetically distinguishable whole bone marrow (BM) populations. Donor BM was competed against carrier BM that was coinjected or injected 3 or 4 days later. Radioprotection with delayed carrier injection also was examined by performing the initial HSC transplantation with Hoechst(lo) side population (SP) cells. SP HSC incubated with cytokines and BM stroma to stimulate cell cycling before transplantation also were tested using coinjection or delayed carrier administration. RESULTS: Delayed introduction of carrier whole BM increased peripheral expansion of donor whole BM, freshly isolated HSC, or cytokine-stimulated HSC compared to coinjection with carrier cells. A 3-day delay in carrier administration maintained radioprotection in 100% of lethally irradiated recipients of highly enriched HSC, whereas a 4-day delay did not rescue these recipients from death. When recipients are rescued, recovering host marrow can compete against donor HSC unless sufficient donor cells are injected. CONCLUSIONS: Delayed introduction of carrier BM significantly increases donor HSC engraftment and peripheral expansion by reducing competition in the host. Competition by a coinjected carrier cell population or recovery of host marrow significantly reduces the therapeutic efficacy of normal or in vitro manipulated donor HSC.
Assuntos
Transplante de Medula Óssea/métodos , Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas/métodos , Transplante Homólogo/métodos , Animais , Benzimidazóis/análise , Divisão Celular , Linhagem da Célula , Modelos Animais de Doenças , Corantes Fluorescentes/análise , Glucuronidase/deficiência , Glucuronidase/genética , Glucuronidase/fisiologia , Células-Tronco Hematopoéticas/química , Células-Tronco Hematopoéticas/classificação , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Mutantes , Mucopolissacaridose VII/genética , Mucopolissacaridose VII/terapia , Tolerância a Radiação , Fatores de TempoRESUMO
To block development of progressive childhood diseases, in utero transplantation (IUTx) requires immediate and significant donor peripheral blood (PB) cell amplification. To date, negligible and nontherapeutic donor PB cell levels have been observed postnatally, except in patients with immunodeficiency diseases. Donor cell fate in utero still is not clear. Ease of identifying and quantifying beta-glucuronidase (GUSB)-expressing donor cells in GUSB-null mucopolysaccharidosis type VII (MPSVII) mouse recipients allowed us to evaluate temporal donor cell engraftment and amplification post-IUTx. Like humans, MPSVII mice are unable to catabolize lysosomal glycosaminoglycans and progressively develop severe storage disease unless they are treated early in life.IUTx recipients were nonablated MPSVII fetuses and genetically stem cell-deficient, and hence myeloablated, W(41)/W(41) MPSVII fetuses. Donor GUSB+ cells were identified and counted in histochemical tissue sections. Quantitative results were confirmed by flow cytometry, enzyme analysis, and histopathology. Whereas GUSB+ cells engraft in most tissues in utero, significant amplification does not occur until the first postnatal week in the nonablated MPSVII hosts. In contrast, genetically myeloablated MPSVII recipients display widely distributed donor cell replacement accompanied by extensive amplification in utero. In both models, storage is alleviated in adult tissues with significant donor cell repopulation. To become therapeutic, IUTx must overcome the limitations of donor cell expansion in the highly competitive fetal environment. Fortunately, nonablative mechanisms to amplify cells in utero are coming on line.
Assuntos
Doenças Fetais/terapia , Glucuronidase/análise , Mucopolissacaridose VII/terapia , Transplante de Células-Tronco/métodos , Animais , Contagem de Células , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fatores de TempoRESUMO
Chromosomal translocations that disrupt transcriptional regulators are frequently involved in the etiology of leukemia. To gain an understanding of the normal and pathologic roles of these transcriptional regulators, both gain- and loss-of-function mutations have been examined in the context of steady-state hematopoiesis. These studies have identified a remarkable number of genes whose loss-of-function phenotype includes a perturbation of hematopoietic stem cell (HSC) proliferation. As more of these models are generated and analyzed using commonly available tools, the regulatory pathways that control HSC quiescence and proliferation are becoming clearer. An emerging theme is that leukemia-associated transcriptional regulators coordinate the balance of proliferation and quiescence within the HSC pool by modulating the number and frequency of cells transiting the cell cycle. Uncoupling proliferation from differentiation by the aberrant generation of chimeric oncogenes that retain some, but not all of the attributes of the original transcription factor is likely to be an important step during leukemogenesis.
Assuntos
Células-Tronco Hematopoéticas/patologia , Leucemia/patologia , Animais , Ciclo Celular , Proliferação de Células , Homeostase , Humanos , Mutação/genéticaRESUMO
The Mixed Lineage Leukemia (MLL) gene is essential for embryonic hematopoietic stem cell (HSC) development, but its role during adult hematopoiesis is unknown. Using an inducible knockout model, we demonstrate that Mll is essential for the maintenance of adult HSCs and progenitors, with fatal bone marrow failure occurring within 3 weeks of Mll deletion. Mll-deficient cells are selectively lost from mixed bone marrow chimeras, demonstrating their failure to self-renew even in an intact bone marrow environment. Surprisingly, HSCs lacking Mll exhibit ectopic cell-cycle entry, resulting in the depletion of quiescent HSCs. In contrast, Mll deletion in myelo-erythroid progenitors results in reduced proliferation and reduced response to cytokine-induced cell-cycle entry. Committed lymphoid and myeloid cells no longer require Mll, defining the early multipotent stages of hematopoiesis as Mll dependent. These studies demonstrate that Mll plays selective and independent roles within the hematopoietic system, maintaining quiescence in HSCs and promoting proliferation in progenitors.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Alelos , Animais , Medula Óssea/patologia , Contagem de Células , Morte Celular , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Proliferação de Células , Sobrevivência Celular , Quimera , Células Precursoras Eritroides/citologia , Éxons/genética , Células-Tronco Hematopoéticas/citologia , Homeostase , Humanos , Camundongos , Células Mieloides/citologia , Células Progenitoras Mieloides/citologia , Deleção de Sequência , Homologia de Sequência de AminoácidosRESUMO
A significant number of nonmalignant, progressive childhood disorders respond to bone marrow transplantation (BMT). Toxic myeloablative pretreatment regimens, graft failure, and graft-vs-host disease complicate the utility of BMT for neonatal treatment. We recently demonstrated high-dose BMT in neonatal animals enables chimeric engraftment without toxic myeloablation. Reagents that block T cell costimulation (anti-CD40L mAb and/or CTLA-4Ig) establish tolerant allogeneic engraftment in adult recipients. Donor lymphocyte infusion (DLI) re-establishes failing grafts and treats malignant relapse via a graft-vs-leukemia response. In this study, we tested the hypothesis that combining these approaches would allow tolerant allogeneic engraftment devoid of myeloablation in neonatal normal and mutant mice with lysosomal storage disease. Tolerant chimeric allogeneic engraftment was achieved before DLI only in the presence of both anti-CD40L mAb and CTLA-4Ig. DLI amplified allografts to full donor engraftment long-term. DLI-treated mice either maintained long-term tolerance or developed late-onset chronic graft-vs-host disease. This combinatorial approach provides a nontoxic method to establish tolerant allogeneic engraftment for treatment of progressive childhood diseases.
Assuntos
Animais Recém-Nascidos/imunologia , Anticorpos Bloqueadores/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Transplante de Medula Óssea/imunologia , Ligante de CD40/imunologia , Imunoconjugados/administração & dosagem , Ativação Linfocitária/imunologia , Condicionamento Pré-Transplante/métodos , Abatacepte , Animais , Animais Recém-Nascidos/genética , Transplante de Medula Óssea/métodos , Transplante de Medula Óssea/patologia , Ligante de CD40/fisiologia , Células Cultivadas , Quimera/imunologia , Doença Crônica , Quimioterapia Combinada , Feminino , Facilitação Imunológica de Enxerto/métodos , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Antígenos H-2/genética , Humanos , Tolerância Imunológica/genética , Ativação Linfocitária/genética , Transfusão de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Mutantes , Transplante HomólogoRESUMO
Lysosomal storage diseases (LSD) respond to bone marrow (BM) transplantation when donor-derived cells deliver needed enzyme. Hypothetically, the ubiquitous resident macrophages (MPhi) are the primary delivery vehicle of therapeutic protein. In mucopolysaccharidosis type VII (MPS VII) mice with LSD, transplanted mature MPhi reduce undegraded glycosaminoglycans (GAG) in the lysosome but are incapable of self-renewal, leading to return of storage after 1 month. We show here that a population of early BM-derived myeloid progenitors devoid of long-term hematopoietic stem cells (LT-HSC) engrafted MPS VII BM, released monocytes into peripheral blood (PBL), and engrafted tissues at known sites of resident MPhi. These primitive Mac-1- cells were sorted from normal whole BM and were defined by ER-MP12hi20-58med/hi labeling. Lysosomal storage was reduced in liver, spleen, thymus, heart, kidney, and bone. Cells persisted for 3 months, suggesting self-renewal capacity or a long half-life. Cells sorted from BM by ER-MP12-20hi marker expression (which are maturer myeloid cells that express Mac-1) engrafted tissues instead of BM and quantitatively repopulated less than cells derived from the ER-MP12hi20-58med/hi population. Also, reduction of lysosomal storage was variable and generally less when compared to that following transplantation of immature ER-MP12hi20-58med/hi cells. We conclude that primitive myeloid progenitors are more therapeutic for LSD than mature myeloid cells due to their greater longevity and increased capacity to seed tissues. The ability of cells derived from these primitive precursors to seed deep within tissues make them excellent candidates for both cellular therapy and gene transfer techniques to cure a wide range of metabolic diseases.