RESUMEN
Given the relatively long life of stem cells (SCs), efficient mechanisms of quality control to balance cell survival and resistance to external and internal stress are required. Our objective was to test the relevance of cell quality control mechanisms for SCs maintenance, differentiation and resistance to cell death. We compared cell quality control in P19 stem cells (P19SCs) before and after differentiation (P19dCs). Differentiation of P19SCs resulted in alterations in parameters involved in cell survival and protein homeostasis, including the redox system, cardiolipin and lipid profiles, unfolded protein response, ubiquitin-proteasome and lysosomal systems, and signaling pathways controlling cell growth. In addition, P19SCs pluripotency was correlated with stronger antioxidant protection, modulation of apoptosis, and activation of macroautophagy, which all contributed to preserve SCs quality by increasing the threshold for cell death activation. Furthermore, our findings identify critical roles for the PI3K-AKT-MTOR pathway, as well as autophagic flux and apoptosis regulation in the maintenance of P19SCs pluripotency and differentiation potential.Abbreviations: 3-MA: 3-methyladenine; AKT/protein kinase B: thymoma viral proto-oncogene; AKT1: thymoma viral proto-oncogene 1; ATG: AuTophaGy-related; ATF6: activating transcription factor 6; BAX: BCL2-associated X protein; BBC3/PUMA: BCL2 binding component 3; BCL2: B cell leukemia/lymphoma 2; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; CASP3: caspase 3; CASP8: caspase 8; CASP9: caspase 9; CL: cardiolipin; CTSB: cathepsin B; CTSD: cathepsin D; DDIT3/CHOP: DNA-damage inducible transcript 3; DNM1L/DRP1: dynamin 1-like; DRAM1: DNA-damage regulated autophagy modulator 1; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; EIF2S1/eIF2α: eukaryotic translation initiation factor 2, subunit alpha; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; ESCs: embryonic stem cells; KRT8/TROMA-1: cytokeratin 8; LAMP2A: lysosomal-associated membrane protein 2A; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NANOG: Nanog homeobox; NAO: 10-N-nonyl acridine orange; NFE2L2/NRF2: nuclear factor, erythroid derived 2, like 2; OPA1: OPA1, mitochondrial dynamin like GTPase; P19dCs: P19 differentiated cells; P19SCs: P19 stem cells; POU5F1/OCT4: POU domain, class 5, transcription factor 1; PtdIns3K: phosphatidylinositol 3-kinase; RA: retinoic acid; ROS: reactive oxygen species; RPS6KB1/p70S6K: ribosomal protein S6 kinase, polypeptide 1; SCs: stem cells; SOD: superoxide dismutase; SHC1-1/p66SHC: src homology 2 domain-containing transforming protein C1, 66 kDa isoform; SOX2: SRY (sex determining region Y)-box 2; SQSTM1/p62: sequestosome 1; SPTAN1/αII-spectrin: spectrin alpha, non-erythrocytic 1; TOMM20: translocase of outer mitochondrial membrane 20; TRP53/p53: transformation related protein 53; TUBB3/betaIII-tubulin: tubulin, beta 3 class III; UPR: unfolded protein response; UPS: ubiquitin-proteasome system.
Asunto(s)
Diferenciación Celular , Células Madre Neoplásicas/patología , Factor de Transcripción Activador 6/metabolismo , Animales , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Cardiolipinas/metabolismo , Inhibidores de Caspasas/farmacología , Compartimento Celular , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Endosomas/metabolismo , Endosomas/ultraestructura , Factor 2 Eucariótico de Iniciación/metabolismo , Lípidos/química , Lisosomas/metabolismo , Lisosomas/ultraestructura , Ratones , Dinámicas Mitocondriales/efectos de los fármacos , Mitofagia/efectos de los fármacos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/ultraestructura , Fosfatidilinositol 3-Quinasas/metabolismo , Proteolisis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
Pancreas transplantation is a therapeutic option for the management of complicated Type 1 diabetes mellitus. While standard protocols include use of induction therapy followed by maintenance immunosuppression, the amount, frequency and duration of induction treatment has not been clearly defined. While the effect of various induction regimens on lymphocytes has been demonstrated, a prospective immune monitoring approach is not widely used to determine "immunological titration" of immunosuppression. In this study, we analyzed a patient cohort with pancreas after kidney transplantation and measured a wide range of quantitative and functional T and B cell parameters to identify those that would provide greatest value in personalized prospective immune assessment and design of immunosuppression. While there were significant quantitative differences observed in the 2 groups of PAK patients for various lymphocyte subsets, the notable observation was that lymphocyte subset quantitation was uninformative with regard to T cell function. Patients with normal lymphocyte counts had impaired T cell functional responses and vice versa. The use of immune monitoring to determine optimal IS regimens needs to be studied further to facilitate personalized management of immunosuppression with reduced risk of allograft rejection in PAK, and limited morbidity and mortality related to infection and malignancy.
