RESUMEN
Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure disorder in which pure red blood cell aplasia is associated with physical malformations and a predisposition to cancer. Twentyfive percent of patients with DBA have mutations in a gene encoding ribosomal protein S19 (RPS19). Our previous proof-of-concept studies demonstrated that DBA phenotype could be successfully treated using lentiviral vectors in Rps19-deficient DBA mice. In our present study, we developed a clinically applicable single gene, self-inactivating lentiviral vector, containing the human RPS19 cDNA driven by the human elongation factor 1a short promoter, which can be used for clinical gene therapy development for RPS19-deficient DBA. We examined the efficacy and safety of the vector in a Rps19-deficient DBA mouse model and in human primary RPS19-deficient CD34+ cord blood cells. We observed that transduced Rps19-deficient bone marrow cells could reconstitute mice long-term and rescue the bone marrow failure and severe anemia observed in Rps19-deficient mice, with a low risk of mutagenesis and a highly polyclonal insertion site pattern. More importantly, the vector can also rescue impaired erythroid differentiation in human primary RPS19-deficient CD34+ cord blood hematopoietic stem cells. Collectively, our results demonstrate the efficacy and safety of using a clinically applicable lentiviral vector for the successful treatment of Rps19-deficient DBA in a mouse model and in human primary CD34+ cord blood cells. These findings show that this vector can be used to develop clinical gene therapy for RPS19-deficient DBA patients.
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Anemia de Diamond-Blackfan , Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/metabolismo , Anemia de Diamond-Blackfan/terapia , Animales , Sangre Fetal/metabolismo , Terapia Genética , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Mutación , ARN Interferente Pequeño/genética , Proteínas Ribosómicas/genéticaRESUMEN
Even though hematopoietic stem cells (HSC) are characterized by their ability to self-renew and differentiate, they primarily reside in quiescence. Despite the immense importance of this quiescent state, its maintenance and regulation is still incompletely understood. Schlafen2 (Slfn2) is a cytoplasmic protein known to be involved in cell proliferation, differentiation, quiescence, interferon response, and regulation of the immune system. Interestingly, Slfn2 is highly expressed in primitive hematopoietic cells. In order to investigate the role of Slfn2 in the regulation of HSC we have studied HSC function in the elektra mouse model, where the elektra allele of the Slfn2 gene contains a point mutation causing loss of function of the Slfn2 protein. We found that homozygosity for the elektra allele caused a decrease of primitive hematopoietic compartments in murine bone marrow. We further found that transplantation of elektra bone marrow and purified HSC resulted in a significantly reduced regenerative capacity of HSC in competitive transplantation settings. Importantly, we found that a significantly higher fraction of elektra HSC (as compared to wild-type HSC) were actively cycling, suggesting that the mutation in Slfn2 increases HSC proliferation. This additionally caused an increased amount of apoptotic stem and progenitor cells. Taken together, our findings demonstrate that dysregulation of Slfn2 results in a functional deficiency of primitive hematopoietic cells, which is particularly reflected by a drastically impaired ability to reconstitute the hematopoietic system following transplantation and an increase in HSC proliferation. This study thus identifies Slfn2 as a novel and critical regulator of adult HSC and HSC quiescence.
Asunto(s)
Proteínas de Ciclo Celular , Hematopoyesis , Células Madre Hematopoyéticas , Animales , Ratones , Médula Ósea , Diferenciación Celular/genética , Proliferación Celular , Células Madre Hematopoyéticas/metabolismo , Proteínas de Ciclo Celular/genéticaRESUMEN
Life-long production of blood from hematopoietic stem cells (HSCs) is a process of strict modulation. Intrinsic and extrinsic signals govern fate options like self-renewal - a cardinal feature of HSCs. Bone morphogenetic proteins (BMP) have an established role in embryonic hematopoiesis, but less is known about its functions in adulthood. Previously, SMAD-mediated BMP signaling has been proven dispensable for HSCs. However, the BMP Type II receptor (BMPR-II) is highly expressed in HSCs, leaving the possibility that BMPs function via alternative pathways. Here, we establish that BMP signaling is required for self-renewal of adult HSCs. Through conditional knockout we show that BMPR-II deficient HSCs have impaired self-renewal and regenerative capacity. BMPR-II deficient cells have reduced p38 activation, implying that non-SMAD pathways operate downstream of BMPs in HSCs. Indeed, a majority of primitive hematopoietic cells do not engage in SMAD-mediated responses downstream of BMPs in vivo. Furthermore, deficiency of BMPR-II results in increased expression of TJP1, a known regulator of self-renewal in other stem cells, and knockdown of TJP1 in primitive hematopoietic cells partly rescues the BMPR-II null phenotype. This suggests TJP1 may be a universal stem cell regulator. In conclusion, BMP signaling, in part mediated through TJP1, is required endogenously by adult HSCs to maintain self-renewal capacity and proper resilience of the hematopoietic system during regeneration.
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Proteínas Morfogenéticas Óseas , Transducción de Señal , Animales , Proteínas Morfogenéticas Óseas/genética , Autorrenovación de las Células , Hematopoyesis , Células Madre Hematopoyéticas , RatonesRESUMEN
OBJECTIVES: Total body irradiation (TBI) is commonly used prior to hematopoietic stem cell transplantation (HSCT) in myeloablative conditioning regimens. However, TBI may be replaced by total marrow irradiation (TMI) at centres with access to Helical TomoTherapy, a modality that has the advantage of delivering intensity-modulated radiotherapy to long targets such as the entire bone marrow compartment. Toxicity after organ sparing TMI prior to HSCT has not previously been reported compared to TBI or with regard to engraftment data. METHODS: We conducted a prospective observational study on 37 patients that received organ sparing TMI prior to HSCT and compared this cohort to retrospective data on 33 patients that received TBI prior to HSCT. RESULTS: The 1-year graft-versus-host disease-free, relapse-free survival (GRFS) was 67.5% for all patients treated with TMI and 80.5% for patients with matched unrelated donor and treated with TMI, which was a significant difference from historical data on TBI patients with a hazard ratio of 0.45 (P = .03) and 0.24 (P < .01). Engraftment with a platelet count over 20 [K/µL] and 50 [K/µL] was significantly shorter for the TMI group, and neutrophil recovery was satisfactory in both treatment cohorts. There was generally a low occurrence of other treatment-related toxicities. CONCLUSIONS: Despite small cohorts, some significant differences were found; TMI as part of the myeloablative conditioning yields a high 1-year GRFS, fast and robust engraftment, and low occurrence of acute toxicity.
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Enfermedad Injerto contra Huésped/patología , Trasplante de Células Madre Hematopoyéticas/métodos , Síndromes Mielodisplásicos/terapia , Trastornos Mieloproliferativos/terapia , Radioterapia de Intensidad Modulada/métodos , Irradiación Corporal Total/métodos , Adolescente , Adulto , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Médula Ósea/efectos de la radiación , Niño , Preescolar , Femenino , Supervivencia de Injerto/fisiología , Enfermedad Injerto contra Huésped/diagnóstico , Enfermedad Injerto contra Huésped/inmunología , Humanos , Masculino , Persona de Mediana Edad , Agonistas Mieloablativos/uso terapéutico , Síndromes Mielodisplásicos/mortalidad , Síndromes Mielodisplásicos/patología , Trastornos Mieloproliferativos/mortalidad , Trastornos Mieloproliferativos/patología , Estudios Prospectivos , Radioterapia de Intensidad Modulada/mortalidad , Análisis de Supervivencia , Acondicionamiento Pretrasplante/métodos , Trasplante Homólogo , Irradiación Corporal Total/mortalidadRESUMEN
The cytokine TGFß1 induces epidermal Langerhans cell (LC) differentiation from human precursors, an effect mediated through BMPR1a/ALK3 signaling, as revealed from ectopic expression and receptor inhibition studies. Whether TGFß1âBMPR1a signaling is required for LC differentiation in vivo remained incompletely understood. We found that TGFß1-deficient mice show defective perinatal expansion and differentiation of LCs. LCs can be identified within the normal healthy human epidermis by anti-BMPR1a immunohistology staining. Deletion of BMPR1a in all (vav+) hematopoietic cells revealed that BMPR1a is required for the efficient TGFß1-dependent generation of CD207+ LC-like cells from CD11c+ intermediates in vitro. Similarly, BMPR1a was required for the optimal induction of CD207 by preformed major histocompatibility complex IIâpositive epidermal resident LC precursors in the steady state. BMPR1a expression is strongly upregulated in epidermal cells in psoriatic lesions, and BMPR1aΔCD11c mice showed a defect in the resolution phase of allergic and psoriatic skin inflammation. Moreover, whereas LCs from these mice expressed CD207, BMPR1a counteracted LC activation and migration from skin explant cultures. Therefore, TGFß1âBMPR1a signaling seems to be required for the efficient induction of CD207 during LC differentiation in the steady state, and bone marrowâderived lesional CD11c+ cells may limit established skin inflammation through enhanced BMPR1a signaling.
Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas de Tipo 1 , Dermatitis , Células de Langerhans , Animales , Antígenos CD/metabolismo , Antígenos de Superficie , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Antígenos CD11 , Antígeno CD11c/metabolismo , Diferenciación Celular , Dermatitis/metabolismo , Epidermis/metabolismo , Inflamación/metabolismo , Células de Langerhans/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Lectinas de Unión a Manosa/metabolismo , RatonesRESUMEN
The emerging notion of hematopoietic stem and progenitor cells (HSPCs) as a low-primed cloud without sharply demarcated gene expression programs raises the question on how cellular-fate options emerge and at which stem-like stage lineage priming is initiated. Here, we investigate single-cell chromatin accessibility of Lineage-, cKit+, and Sca1+ (LSK) HSPCs spanning the early differentiation landscape. Application of a signal-processing algorithm to detect transition points corresponding to massive alterations in accessibility of 571 transcription factor motifs reveals a population of LSK FMS-like tyrosine kinase 3 (Flt3)intCD9high cells that concurrently display stem-like and lineage-affiliated chromatin signatures, pointing to a simultaneous gain of both lympho-myeloid and megakaryocyte-erythroid programs. Molecularly and functionally, these cells position between stem cells and committed progenitors and display multi-lineage capacity in vitro and in vivo but lack self-renewal activity. This integrative molecular analysis resolves the heterogeneity of cells along hematopoietic differentiation and permits investigation of chromatin-mediated transition between multipotency and lineage restriction.
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Cromatina , Células Madre Hematopoyéticas , Diferenciación Celular , Linaje de la Célula , Cromatina/metabolismo , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , MegacariocitosRESUMEN
Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes coding for ribosomal proteins. Among these genes, the ribosomal protein S19 (RPS19) gene is the most frequently mutated. Previously, a mouse model deficient in RPS19 was developed by our laboratory, which recapitulates the hematopoietic disease phenotype by manifesting pathologic features and clinical symptoms of DBA. Characterization of this model revealed that chronic RPS19 deficiency leads to exhaustion of hematopoietic stem cells and subsequent bone marrow (BM) failure. In this study, we evaluated a nonmyeloablative conditioning protocol for BM transplants in RPS19-deficient mice by transplanting wild-type BM cells to RPS19-deficient recipients given no conditioning or sublethal doses of irradiation before transplant. We describe full correction of the hematopoietic phenotype in mice given sublethal doses of irradiation, as well as in animals completely devoid of any preceding irradiation. In comparison, wild-type animals receiving the same preconditioning regimen and number of transplanted cells exhibited significantly lower engraftment levels. Thus, robust engraftment and repopulation of transplanted cells can be achieved in reduced-intensity conditioned RPS19-deficient recipients. As gene therapy studies with autologous gene-corrected hematopoietic stem cells are emerging, we propose the results described here can guide determination of the level of conditioning for such a protocol in RPS19-deficient DBA. On the basis of our findings, a relatively mild conditioning strategy would plausibly be sufficient to achieve sufficient levels of engraftment and clinical success.
Asunto(s)
Anemia de Diamond-Blackfan/metabolismo , Trasplante de Médula Ósea , Aloinjertos , Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/patología , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Noqueados , Proteínas Ribosómicas/deficiencia , Proteínas Ribosómicas/metabolismo , Acondicionamiento PretrasplanteRESUMEN
Gaucher disease type 1 (GD1) is an inherited lysosomal disorder with multisystemic effects in patients. Hallmark symptoms include hepatosplenomegaly, cytopenias, and bone disease with varying degrees of severity. Mutations in a single gene, glucosidase beta acid 1 (GBA1), are the underlying cause for the disorder, resulting in insufficient activity of the enzyme glucocerebrosidase, which in turn leads to a progressive accumulation of the lipid component glucocerebroside. In this study, we treat mice with signs consistent with GD1, with hematopoietic stem/progenitor cells transduced with a lentiviral vector containing an RNA transcript that, after reverse transcription, results in codon-optimized cDNA that, upon its integration into the genome encodes for functional human glucocerebrosidase. Five months after gene transfer, a highly significant reduction in glucocerebroside accumulation with subsequent reversal of hepatosplenomegaly, restoration of blood parameters, and a tendency of increased bone mass and density was evident in vector-treated mice compared to non-treated controls. Furthermore, histopathology revealed a prominent reduction of Gaucher cell infiltration after gene therapy. The vector displayed an oligoclonal distribution pattern but with no sign of vector-induced clonal dominance and a typical lentiviral vector integration profile. Cumulatively, our findings support the initiation of the first clinical trial for GD1 using the lentiviral vector described here.
RESUMEN
The fate options of hematopoietic stem cells (HSCs) include self-renewal, differentiation, migration, and apoptosis. HSCs self-renewal divisions in stem cells are required for rapid regeneration during tissue damage and stress, but how precisely intracellular calcium signals are regulated to maintain fate options in normal hematopoiesis is unclear. S100A6 knockout (KO) HSCs have reduced total cell numbers in the HSC compartment, decreased myeloid output, and increased apoptotic HSC numbers in steady state. S100A6KO HSCs had impaired self-renewal and regenerative capacity, not responding to 5-Fluorouracil. Our transcriptomic and proteomic profiling suggested that S100A6 is a critical HSC regulator. Intriguingly, S100A6KO HSCs showed decreased levels of phosphorylated Akt (p-Akt) and Hsp90, with an impairment of mitochondrial respiratory capacity and a reduction of mitochondrial calcium levels. We showed that S100A6 regulates intracellular and mitochondria calcium buffering of HSC upon cytokine stimulation and have demonstrated that Akt activator SC79 reverts the levels of intracellular and mitochondrial calcium in HSC. Hematopoietic colony-forming activity and the Hsp90 activity of S100A6KO are restored through activation of the Akt pathway. We show that p-Akt is the prime downstream mechanism of S100A6 in the regulation of HSC self-renewal by specifically governing mitochondrial metabolic function and Hsp90 protein quality.
RESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Pigment epithelium derived factor (PEDF), a ubiquitously expressed 50 kDa secreted glycoprotein, was recently discovered to regulate self-renewal of neural stem cells and have a supportive effect on human embryonic stem cell growth. Here, we analyzed expression of PEDF in the murine hematopoietic stem cell (HSC) compartments and found that PEDF is highly expressed in primary long-term HSCs. Therefore, we characterized the hematopoietic system in a knockout mouse model for PEDF and using this model we surprisingly found that PEDF is dispensable for HSC regulation. PEDF knockout mice exhibit normal hematopoiesis in steady state conditions and the absence of PEDF lead to normal regeneration capacity in a serial competitive transplantation setting. Additionally, PEDF-deficient cells exhibit unaltered lineage distribution upon serial transplantations. When human cord blood stem and progenitor cells were cultured in media supplemented with recombinant PEDF they did not show changes in growth potential. Taken together, we report that PEDF is not a critical regulatory factor for HSC function during regeneration in vivo or growth of human stem/progenitor cells in vitro.
Asunto(s)
Proteínas del Ojo/metabolismo , Hematopoyesis , Células Madre Hematopoyéticas/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Serpinas/metabolismo , Animales , Células Cultivadas , Proteínas del Ojo/genética , Células Madre Hematopoyéticas/citología , Humanos , Ratones , Ratones Endogámicos C57BL , Factores de Crecimiento Nervioso/genética , Serpinas/genéticaRESUMEN
Impulse control disorders (ICDs) are reported in Parkinson's disease (PD) in association with dopaminergic treatment. Approximately 25 % of patients with ICDs have multiple co-occurring ICDs (i.e. more than one diagnosed ICD). The extent to which dopaminergic neurotransmission in PD patients with multiple ICDs differs from those with only one diagnosed ICD is unknown. The aims of this study are: (1) to investigate dopamine neurotransmission in PD patients diagnosed with multiple ICDs, single ICDs and non-ICD controls in response to reward-related visual cues using positron emission tomography with (11)C-raclopride. (2) to compare clinical features of the above three groups. PD individuals with mulitple ICDs (n = 10), single ICD (n = 7) and no ICDs (n = 9) were recruited and underwent two positron emission tomography (PET) scans with (11)C-raclopride: one where they viewed neutral visual cues and the other where they viewed a range of visual cues related to different rewards. Individuals with both multiple ICDs and single ICDs showed significantly greater ventral striatal dopamine release compared to non-ICD PD individuals in response to reward cues, but the two ICD groups did not differ from each other in the extent of dopamine release. Subjects with multiple ICDs were, however, significantly more depressed, and had higher levels of impulsive sensation-seeking compared to subjects with single ICDs and without ICDs. This is the first study to compare dopamine neurotransmission using PET neuroimaging in PD subjects with multiple vs. single ICDs. Our results suggest that striatal dopamine neurotransmission is not directly related to the co-occurrence of ICDs in PD, potentially implicating non-dopaminergic mechanisms linked to depression; and suggest that physicians should be vigilant in managing depression in PD patients with ICDs.
Asunto(s)
Cuerpo Estriado/diagnóstico por imagen , Señales (Psicología) , Trastornos Disruptivos, del Control de Impulso y de la Conducta , Enfermedad de Parkinson/complicaciones , Tomografía de Emisión de Positrones , Recompensa , Análisis de Varianza , Isótopos de Carbono/farmacocinética , Cuerpo Estriado/efectos de los fármacos , Trastornos Disruptivos, del Control de Impulso y de la Conducta/diagnóstico por imagen , Trastornos Disruptivos, del Control de Impulso y de la Conducta/etiología , Trastornos Disruptivos, del Control de Impulso y de la Conducta/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/radioterapia , Escalas de Valoración Psiquiátrica , Racloprida/farmacocinéticaRESUMEN
BACKGROUND: Problematic Internet use (PIU) has been associated with impulse control disorders (ICDs), and postulated to share characteristics of a behavioral addiction with both impulsive and compulsive features. However, Internet use has not been previously systematically studied in Parkinson's disease. AIM: We explore Internet use in PD patients with and without ICDs and matched healthy controls. We hypothesize that the PD-ICD patients will spend more time on the Internet, accessing websites related to their ICDs, compared with PD patients without ICDs and healthy volunteers. METHODS: Our study is the first to systematically explore problematic Internet use in patients with PD, with and without ICDs. Twenty-nine PD patients with ICDs, twenty PD patients without ICDs and nineteen healthy controls were recruited. All participants endorsed using the Internet for non-essential purposes. They underwent a semi-structured interview and completed questionnaires including the Yale-Brown Obsessive Compulsive Scale adapted for Internet use (Y-BOCS-Internet). RESULTS: PD-ICD patients scored significantly higher on the Y-BOCS-Internet than the PD-control and HV groups (PD-ICD: 13.69; PD-control: 5.42; HV: 4.70; p < 0.0001). Compared to PD controls and HV groups, the PD-ICD group spent more time on the Internet (p = 0.0001), described significantly more effort to resist Internet use (p = 0.0002), thoughts about Internet use (p < 0.0001) and its interference with their life functioning (p = 0.0025). DISCUSSION: Our results suggest that PD patients with ICDs have a relative increased tendency towards excessive Internet use compared to those without ICDs and healthy controls. Clinicians should actively screen for excessive Internet use in patients with ICDs.