RESUMEN
Oncogenic NRAS mutations are frequently identified in human myeloid leukemias. In mice, expression of endogenous oncogenic Nras (Nras(G12D/+)) in hematopoietic cells leads to expansion of myeloid progenitors, increased long-term reconstitution of bone marrow cells, and a chronic myeloproliferative neoplasm (MPN). However, acute expression of Nras(G12D/+) in a pure C57BL/6 background does not induce hyperactivated granulocyte macrophage colony-stimulating factor signaling or increased proliferation in myeloid progenitors. It is thus unclear how Nras(G12D/+) signaling promotes leukemogenesis. Here, we show that hematopoietic stem cells (HSCs) expressing Nras(G12D/+) serve as MPN-initiating cells. They undergo moderate hyperproliferation with increased self-renewal. The aberrant Nras(G12D/+) HSC function is associated with hyperactivation of ERK1/2 in HSCs. Conversely, downregulation of MEK/ERK by pharmacologic and genetic approaches attenuates the cycling of Nras(G12D/+) HSCs and prevents the expansion of Nras(G12D/+) HSCs and myeloid progenitors. Our data delineate critical mechanisms of oncogenic Nras signaling in HSC function and leukemogenesis.
Asunto(s)
GTP Fosfohidrolasas/fisiología , Células Madre Hematopoyéticas/patología , Leucemia Mielomonocítica Crónica/genética , Leucemia Mielomonocítica Crónica/patología , MAP Quinasa Quinasa 1/metabolismo , Proteínas de la Membrana/fisiología , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mutación/genética , Animales , Citometría de Flujo , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Mielomonocítica Crónica/metabolismo , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Ratones , Ratones Transgénicos , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Fosforilación , Transducción de SeñalRESUMEN
Human myogenic progenitors can be derived from pluripotent stem cells (PSCs) for use in modeling natural and pathological myogenesis, as well as treating muscle diseases. Transgene-free methods of deriving myogenic progenitors from different PSC lines often produce mixed populations that are heterogeneous in myogenic differentiation potential, yet detailed and accurate characterization of human PSC-derived myogenic progenitors remains elusive in the field. The isolation and purification of human PSC-derived myogenic progenitors is thus an important methodological consideration when we investigate the properties and behaviors of these cells in culture. We previously reported a transgene-free, serum-free floating sphere culture method for the derivation of myogenic progenitors from human PSCs. In this study, we first performed comprehensive cell surface protein profiling of the sphere culture cells through the screening of 255 antibodies. Next, we used magnetic activated cell sorting and enriched the cells according to the expression of specific surface markers. The ability of muscle differentiation in the resulting cells was characterized by immunofluorescent labeling and quantification of positively stained cells. Our results revealed that myotube-forming cells resided in the differentiated cultures of CD29+, CD56+, CD271+, and CD15- fractions, while thick and multinucleated myotubes were identified in the differentiated cultures from CD9+ and CD146+ fractions. We found that PAX7 localization to the nucleus correlates with myotube-forming ability in these sorted populations. We also demonstrated that cells in unsorted, CD271+, and CD15- fractions responded differently to cryopreservation and prolonged culture expansion. Lastly, we showed that CD271 expression is essential for terminal differentiation of human PSC-derived myogenic progenitors. Taken together, these cell surface proteins are not only useful markers to identify unique cellular populations in human PSC-derived myogenic progenitors but also functionally important molecules that can provide valuable insight into human myogenesis.
RESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease in which patients gradually become paralyzed due to loss of motor function. Many genetically inheritable mutations have been linked to ALS; however, the majority of ALS patients are considered sporadic. Therefore, there is a need for a common therapy that is effective for all ALS patients. Although there is evidence of the disease beginning in the periphery at the neuromuscular junction (NMJ), the specific processes involved in skeletal muscle and at the NMJ are still largely unknown. To study common disease mechanisms in ALS skeletal muscle, we performed RNA sequencing of skeletal myocytes differentiated from induced pluripotent stem cells (iPSCs) derived from familial ALS (with C9ORF72, SOD1, or TARDBP mutations) and sporadic ALS patients. Compared to healthy control lines, the myocytes from all ALS lines showed downregulation of four genes: BET1L, DCX, GPC3, and HNRNPK. We next measured the expression levels of these four genes in hind limb muscle samples from a rat model of familial ALS (SOD1G93A transgenic) and found that only the Bet1L gene, which encodes Bet1 Golgi Vesicular Membrane Trafficking Protein Like, was commonly downregulated. Bet1L protein appeared to be localized to the basal lamina of the NMJ, with decreased expression over time in SOD1G93A transgenic rats. Importantly, the expression levels began to decrease early in the disease process. Our results indicate that loss of Bet1L at the NMJ could be of interest for better understanding ALS disease progression.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Perfilación de la Expresión Génica/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Unión Neuromuscular/metabolismo , Proteínas Qc-SNARE/deficiencia , Adulto , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Diferenciación Celular/fisiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Unión Neuromuscular/patología , Proteínas Qc-SNARE/genética , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Análisis de Secuencia de ARN/métodosRESUMEN
Skeletal muscle progenitor cells (SMPCs), also called myogenic progenitors, have been studied extensively in recent years because of their promising therapeutic potential to preserve and recover skeletal muscle mass and function in patients with cachexia, sarcopenia, and neuromuscular diseases. SMPCs can be utilized to investigate the mechanisms of natural and pathological myogenesis via in vitro modeling and in vivo experimentation. While various types of SMPCs are currently available from several sources, human pluripotent stem cells (PSCs) offer an efficient and cost-effective method to derive SMPCs. As human PSC-derived cells often display varying heterogeneity in cell types, cell enrichment using cell surface markers remains a critical step in current procedures to establish a pure population of SMPCs. Here we summarize the cell surface markers currently being used to detect human SMPCs, describing their potential application for characterizing, identifying and isolating human PSC-derived SMPCs. To date, several positive and negative markers have been used to enrich human SMPCs from differentiated PSCs by cell sorting. A careful analysis of current findings can broaden our understanding and reveal potential uses for these surface markers with SMPCs.
RESUMEN
Overactive RAS signaling is prevalent in juvenile myelomonocytic leukemia (JMML) and the myeloproliferative variant of chronic myelomonocytic leukemia (MP-CMML) in humans, and both are refractory to conventional chemotherapy. Conditional activation of a constitutively active oncogenic Nras (NrasG12D/G12D) in murine hematopoietic cells promotes an acute myeloproliferative neoplasm (MPN) that recapitulates many features of JMML and MP-CMML. We found that NrasG12D/G12D-expressing HSCs, which serve as JMML/MP-CMML-initiating cells, show strong hyperactivation of ERK1/2, promoting hyperproliferation and depletion of HSCs and expansion of downstream progenitors. Inhibition of the MEK pathway alone prolonged the presence of NrasG12D/G12D-expressing HSCs but failed to restore their proper function. Consequently, approximately 60% of NrasG12D/G12D mice treated with MEK inhibitor alone died within 20 weeks, and the remaining animals continued to display JMML/MP-CMML-like phenotypes. In contrast, combined inhibition of MEK and JAK/STAT signaling, which is commonly hyperactivated in human and mouse CMML, potently inhibited human and mouse CMML cell growth in vitro, rescued mutant NrasG12D/G12D-expressing HSC function in vivo, and promoted long-term survival without evident disease manifestation in NrasG12D/G12D animals. These results provide a strong rationale for further exploration of combined targeting of MEK/ERK and JAK/STAT in treating patients with JMML and MP-CMML.