RESUMEN
Generating engraftable skeletal muscle progenitor cells is a promising cell therapy approach to treating degenerating muscle diseases. Pluripotent stem cell (PSC) is an ideal cell source for cell therapy because of its unlimited proliferative capability and potential to differentiate into multiple lineages. Approaches such as ectopic overexpression of myogenic transcription factors and growth factors-directed monolayer differentiation, while able to differentiate PSCs into the skeletal myogenic lineage in vitro, are limited in producing muscle cells that reliably engraft upon transplantation. Here we present a novel method to differentiate mouse PSCs into skeletal myogenic progenitors without genetic modification or monolayer culture. We make use of forming a teratoma, in which skeletal myogenic progenitors can be routinely obtained. We first inject mouse PSCs into the limb muscle of an immuno-compromised mouse. Within 3-4 weeks, α7-integrin+ VCAM-1+ skeletal myogenic progenitors are purified by fluorescent-activated cell sorting. We further transplant these teratoma-derived skeletal myogenic progenitors into dystrophin-deficient mice to assess engraftment efficiency. This teratoma formation strategy is capable of generating skeletal myogenic progenitors with high regenerative potency from PSCs without genetic modifications or growth factors supplementation.
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Células Madre Pluripotentes , Células Satélite del Músculo Esquelético , Teratoma , Ratones , Animales , Células Satélite del Músculo Esquelético/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Diferenciación Celular , Péptidos y Proteínas de Señalización Intercelular , Teratoma/etiología , Músculo Esquelético/metabolismo , Desarrollo de MúsculosRESUMEN
Insulin-producing cells derived from induced pluripotent stem cells (iPSCs) are promising candidates for ß cell replacement in type 1 diabetes. However, the risk of teratoma formation due to residual undifferentiated iPSCs contaminating the differentiated cells is still a critical concern for clinical application. Here, we hypothesized that pretreatment of iPSC-derived insulin-producing cells with an anti-CD30 antibody−drug conjugate could prevent in vivo teratoma formation by selectively killing residual undifferentiated cells. CD30 is expressed in all human iPSCs clones tested by flow cytometry (n = 7) but not in iPSC-derived ß cells (ißs). Concordantly, anti-CD30 treatment in vitro for 24 h induced a dose-dependent cell death (up to 90%) in human iPSCs while it did not kill ißs nor had an impact on iß identity and function, including capacity to secrete insulin in response to stimuli. In a model of teratoma assay associated with iß transplantation, the pretreatment of cells with anti-CD30 for 24 h before the implantation into NOD-SCID mice completely eliminated teratoma development (0/10 vs. 8/8, p < 0.01). These findings suggest that short-term in vitro treatment with clinical-grade anti-CD30, targeting residual undifferentiated cells, eliminates the tumorigenicity of iPSC-derived ß cells, potentially providing enhanced safety for iPSC-based ß cell replacement therapy in clinical scenarios.
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Antineoplásicos , Inmunoconjugados , Células Madre Pluripotentes Inducidas , Teratoma , Animales , Antineoplásicos/farmacología , Diferenciación Celular , Humanos , Inmunoconjugados/farmacología , Insulina/metabolismo , Antígeno Ki-1/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Teratoma/etiología , Teratoma/metabolismo , Teratoma/prevención & controlRESUMEN
BACKGROUND: Laparoscopic removal of ovarian dermoid cysts has been associated with increased risk for recurrence. OBJECTIVES: To investigate the risk factors associated with recurrence of dermoid cysts. METHODS: We conducted a retrospective review of all women who underwent cystectomy for ovarian dermoid cysts by laparoscopy or laparotomy. At discharge, patients were instructed to undergo a yearly ultrasound exam. A follow-up telephone call was conducted to assess whether an additional surgery for dermoid cysts was required and whether ultrasound recurrence of dermoid cysts was suspected. RESULTS: The study cohort included 102 participants (92 [90.2%] operated by laparoscopy and 10 [9.8%] by laparotomy). The mean follow-up from the index surgery to the interview was 72.1 ± 38.2 months. The rates of recurrent surgery were similar among women who underwent laparoscopic cystectomy compared with laparotomy (5/92 [5.4%] vs. 1/10 [10.0%], respectively; P = 0.5), while the rates of reported ultrasound recurrence were significantly lower in the laparoscopy group compared with the laparotomy group (10/102 [10.9%] vs. 4/10 [40.0%], respectively; P = 0.03). Additional factors including age, cyst diameter, diagnosis of torsion, intraoperative cyst spillage, estimated blood loss, intraperitoneal adhesions, and postoperative fever were not associated with recurrence. CONCLUSIONS: Ultrasound recurrence of dermoid cysts is not uncommon and could be associated with the surgical approach.
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Quiste Dermoide , Laparoscopía , Neoplasias Ováricas , Teratoma , Quiste Dermoide/diagnóstico por imagen , Quiste Dermoide/epidemiología , Femenino , Humanos , Laparoscopía/efectos adversos , Laparotomía/efectos adversos , Neoplasias Ováricas/diagnóstico por imagen , Neoplasias Ováricas/epidemiología , Estudios Retrospectivos , Teratoma/etiología , Teratoma/cirugíaRESUMEN
Human pluripotent stem cell (hPSC)-derived pancreatic progenitors (PPs) can be differentiated into beta-like cells in vitro and in vivo and therefore have therapeutic potential for type 1 diabetes (T1D) treatment. However, the purity of PPs varies across different hPSC lines, differentiation protocols, and laboratories. The uncommitted cells may give rise to non-pancreatic endodermal, mesodermal, or ectodermal derivatives in vivo, hampering the safety of hPSC-derived PPs for clinical applications and their differentiation efficiency in research settings. Recently, proteomics and transcriptomics analyses identified glycoprotein 2 (GP2) as a PP-specific cell surface marker. The GP2-enriched PPs generate higher percentages of beta-like cells in vitro, but their potential in vivo remains to be elucidated. Here, we demonstrate that the GP2-enriched-PPs give rise to all pancreatic cells in vivo, including functional beta-like cells. Remarkably, GP2 enrichment eliminates the risk of teratomas, which establishes GP2 sorting as an effective method for PP purification and safe pancreatic differentiation.
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Células Secretoras de Insulina , Células Madre Pluripotentes , Teratoma , Diferenciación Celular/fisiología , Endodermo , Humanos , Células Secretoras de Insulina/metabolismo , Páncreas , Células Madre Pluripotentes/metabolismo , Teratoma/etiología , Teratoma/metabolismoRESUMEN
Sox2 is a core transcription factor in embryonic stem cells (ESCs), and O-GlcNAcylation is a type of post-translational modification of nuclear-cytoplasmic proteins. Although both factors play important roles in the maintenance and differentiation of ESCs and the serine 248 (S248) and threonine 258 (T258) residues of Sox2 are modified by O-GlcNAcylation, the function of Sox2 O-GlcNAcylation is unclear. Here, we show that O-GlcNAcylation of Sox2 at T258 regulates mouse ESC self-renewal and early cell fate. ESCs in which wild-type Sox2 was replaced with the Sox2 T258A mutant exhibited reduced self-renewal, whereas ESCs with the Sox2 S248A point mutation did not. ESCs with the Sox2 T258A mutation heterologously introduced using the CRISPR/Cas9 system, designated E14-Sox2TA/WT, also exhibited reduced self-renewal. RNA sequencing analysis under self-renewal conditions showed that upregulated expression of early differentiation genes, rather than a downregulated expression of self-renewal genes, was responsible for the reduced self-renewal of E14-Sox2TA/WT cells. There was a significant decrease in ectodermal tissue and a marked increase in cartilage tissue in E14-Sox2TA/WT-derived teratomas compared with normal E14 ESC-derived teratomas. RNA sequencing of teratomas revealed that genes related to brain development had generally downregulated expression in the E14-Sox2TA/WT-derived teratomas. Our findings using the Sox2 T258A mutant suggest that Sox2 T258 O-GlcNAc has a positive effect on ESC self-renewal and plays an important role in the proper development of ectodermal lineage cells. Overall, our study directly links O-GlcNAcylation and early cell fate decisions.
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Autorrenovación de las Células , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Factores de Transcripción SOXB1/metabolismo , Treonina/metabolismo , Alelos , Animales , Diferenciación Celular/genética , Linaje de la Célula , Autorrenovación de las Células/genética , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Edición Génica , Regulación de la Expresión Génica , Glicosilación , Ratones , Mutación , Procesamiento Proteico-Postraduccional , Factores de Transcripción SOXB1/genética , Teratoma/etiología , Teratoma/metabolismo , Teratoma/patologíaAsunto(s)
Canal Anal/anomalías , Meningocele/etiología , Recto/anomalías , Sacro/anomalías , Teratoma/etiología , Adulto , Canal Anal/diagnóstico por imagen , Femenino , Humanos , Recién Nacido , Meningocele/diagnóstico , Meningocele/diagnóstico por imagen , Embarazo , Recto/diagnóstico por imagen , Sacro/diagnóstico por imagen , Teratoma/diagnóstico , Teratoma/diagnóstico por imagen , Tomografía Computarizada por Rayos X/métodos , Ultrasonografía/métodosRESUMEN
Canine induced pluripotent stem cells (ciPSCs) provide a platform for regenerative veterinary medicine, disease modeling, and drug discovery. However, in the conventional method, ciPSCs are maintained using chemically-undefined media containing unknown animal components under on-murine embryonic fibroblast feeder conditions, which were reported to modify cell surface of iPSCs and increases the risk of immune rejection when the cells are transplanted into patients. Moreover, in the conventional method, ciPSCs are mechanically passaged, which requires much time and effort. Therefore, the large-scale expansion of ciPSCs is difficult, which should be resolved for using ciPSCs in clinical application and research. Here, it was shown that StemFit® AK02N and iMatrix-511 could maintain the pluripotency of ciPSCs using conventional culture method. Furthermore, it was demonstrated that the feeder-free and chemically-defined ciPSC culture systems using StemFit® AK02N and iMatrix-511 could stably maintain and allow the easy expansion of ciPSCs generated using N2B27 and StemFit® AK02N, without causing karyotype abnormalities. ciPSCs expressed several pluripotency markers and formed teratomas, including cells derived from three germ layers.
Asunto(s)
Técnicas de Cultivo de Célula , Medios de Cultivo/farmacología , Perros/anatomía & histología , Células Madre Pluripotentes Inducidas/citología , Cultivo Primario de Células/métodos , Animales , Biomarcadores , Adhesión Celular , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula , Células Cultivadas , Células Clonales , Técnicas de Cocultivo , Medios de Cultivo/análisis , Estratos Germinativos/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/trasplante , Cariotipificación , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Proteínas Recombinantes/farmacología , Teratoma/etiología , Teratoma/patologíaRESUMEN
PURPOSE: Report relevance of molecular groups to clinicopathologic features, germline SMARCB1/SMARCA4 alterations (GLA), and survival of children with atypical teratoid rhabdoid tumor (ATRT) treated in two multi-institutional clinical trials. MATERIALS AND METHODS: Seventy-four participants with newly diagnosed ATRT were treated in two trials: infants (SJYC07: age < 3 years; n = 52) and children (SJMB03: age 3-21 years; n = 22), using surgery, conventional chemotherapy (infants), or dose-dense chemotherapy with autologous stem cell rescue (children), and age- and risk-adapted radiotherapy [focal (infants) and craniospinal (CSI; children)]. Molecular groups ATRT-MYC (MYC), ATRT-SHH (SHH), and ATRT-TYR (TYR) were determined from tumor DNA methylation profiles. RESULTS: Twenty-four participants (32%) were alive at time of analysis at a median follow-up of 8.4 years (range, 3.1-14.1 years). Methylation profiling classified 64 ATRTs as TYR (n = 21), SHH (n = 30), and MYC (n = 13), SHH group being associated with metastatic disease. Among infants, TYR group had the best overall survival (OS; P = 0.02). However, outcomes did not differ by molecular groups among infants with nonmetastatic (M0) disease. Children with M0 disease and <1.5 cm2 residual tumor had a 5-year progression-free survival (PFS) of 72.7 ± 12.7% and OS of 81.8 ± 11%. Infants with M0 disease had a 5-year PFS of 39.1 ± 11.5% and OS of 51.8 ± 12%. Those with metastases fared poorly [5-year OS 25 ± 12.5% (children) and 0% (infants)]. SMARCB1 GLAs were not associated with PFS. CONCLUSIONS: Among infants, those with ATRT-TYR had the best OS. ATRT-SHH was associated with metastases and consequently with inferior outcomes. Children with nonmetastatic ATRT benefit from postoperative CSI and adjuvant chemotherapy.
Asunto(s)
Biomarcadores de Tumor , Tumor Rabdoide/diagnóstico , Tumor Rabdoide/etiología , Teratoma/diagnóstico , Teratoma/etiología , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Niño , Preescolar , Variaciones en el Número de Copia de ADN , Metilación de ADN , Diagnóstico Diferencial , Manejo de la Enfermedad , Susceptibilidad a Enfermedades , Femenino , Mutación de Línea Germinal , Humanos , Lactante , Masculino , Mutación , Pronóstico , Tumor Rabdoide/mortalidad , Tumor Rabdoide/terapia , Proteína SMARCB1/genética , Teratoma/mortalidad , Teratoma/terapia , Resultado del TratamientoRESUMEN
Clinical translation of pluripotent stem cell (PSC) derivatives is hindered by the tumorigenic risk from residual undifferentiated cells. Here, we identified salicylic diamines as potent agents exhibiting toxicity to murine and human PSCs but not to cardiomyocytes (CMs) derived from them. Half maximal inhibitory concentrations (IC50) of small molecules SM2 and SM6 were, respectively, 9- and 18-fold higher for human than murine PSCs, while the IC50 of SM8 was comparable for both PSC groups. Treatment of murine embryoid bodies in suspension differentiation cultures with the most effective small molecule SM6 significantly reduced PSC and non-PSC contamination and enriched CM populations that would otherwise be eliminated in genetic selection approaches. All tested salicylic diamines exerted their toxicity by inhibiting the oxygen consumption rate (OCR) in PSCs. No or only minimal and reversible effects on OCR, sarcomeric integrity, DNA stability, apoptosis rate, ROS levels or beating frequency were observed in PSC-CMs, although effects on human PSC-CMs seemed to be more deleterious at higher SM-concentrations. Teratoma formation from SM6-treated murine PSC-CMs was abolished or delayed compared to untreated cells. We conclude that salicylic diamines represent promising compounds for PSC removal and enrichment of CMs without the need for other selection strategies.
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Diferenciación Celular/efectos de los fármacos , Diaminas/farmacología , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Diaminas/química , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Estructura Molecular , Miocitos Cardíacos/citología , Consumo de Oxígeno/efectos de los fármacos , Teratoma/tratamiento farmacológico , Teratoma/etiología , Teratoma/patologíaRESUMEN
Spontaneous testicular teratomas (STTs) derived from primordial germ cells (PGCs) in the mouse embryonic testes predominantly develop in the 129 family inbred strain. Ter (spontaneous mutation) is a single nucleotide polymorphism that generates a premature stop codon of Dead end1 (Dnd1) and increases the incidence of STTs in the 129 genetic background. We previously found that DND1 interacts with NANOS2 or NANOS3 and that these complexes play a vital role in male embryonic germ cells and adult spermatogonia. However, the following are unclear: (a) whether DND1 works with NANOS2 or NANOS3 to regulate teratoma incidence, and (b) whether Ter simply causes Dnd1 loss or produces a short mutant DND1 protein. In the current study, we newly established a conventional Dnd1-knockout mouse line and found that these mice showed phenotypes similar to those of Ter mutant mice in spermatogenesis, oogenesis, and teratoma incidence, with a slight difference in spermiogenesis. In addition, we found that the amount of DND1 in Dnd1+/Ter embryos decreased to half of that in wild-type embryos, while the expression of the short mutant DND1 was not detected. We also found that double mutants for Dnd1 and Nanos2 or Nanos3 showed synergistic increase in the incidence of STTs. These data support the idea that Ter causes Dnd1 loss, leading to an increase in STT incidence, and that DND1 acts with NANOS2 and NANOS3 to regulate the development of teratoma from PGCs in the 129 genetic background. Thus, our results clarify the role of Dnd1 in the development of STTs and provide a novel insight into its pathogenic mechanism.
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Células Germinales Embrionarias/patología , Proteínas de Neoplasias/fisiología , Proteínas de Unión al ARN/metabolismo , Teratoma/etiología , Neoplasias Testiculares/etiología , Testículo/patología , Animales , Células Germinales Embrionarias/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Oogénesis , Proteínas de Unión al ARN/genética , Espermatogénesis , Teratoma/metabolismo , Teratoma/patología , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/patología , Testículo/metabolismoRESUMEN
BACKGROUND: Meningitis is a very rare atypical presenting feature of anti-NMDA receptor encephalitis. In our case report, we describe an unusual clinical presentation of anti-NMDA receptor encephalitis with a biphasic pattern of meningitis followed by encephalitis and discuss potential mechanisms underlying this presentation. We aim to widen the differential diagnosis to be considered in a patient presenting with clinical meningitis and pyrexia. CASE PRESENTATION: This is a case of a 33-year old Caucasian woman who initially presented with a lymphocytic meningitis attributed to a viral infection. She subsequently developed fluctuating consciousness, agitation, visual hallucinations, dyskinetic movements, a generalized tonic-clonic seizure, and autonomic instability. Investigations revealed a diagnosis of anti-NMDA receptor encephalitis secondary to a previously unidentified ovarian teratoma. She made an excellent recovery with immunotherapy and removal of the teratoma. CONCLUSION: Clinicians should consider autoimmune encephalitides in individuals with meningitis, particularly where extensive investigations fail to identify a causative pathogen and there is rapid development of an encephalitic phenotype.
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Encefalitis Antirreceptor N-Metil-D-Aspartato/diagnóstico , Meningitis Viral/diagnóstico , Neoplasias Ováricas/patología , Teratoma/patología , Administración Intravenosa , Adulto , Encefalitis Antirreceptor N-Metil-D-Aspartato/complicaciones , Encefalitis Antirreceptor N-Metil-D-Aspartato/tratamiento farmacológico , Anticuerpos/sangre , Diagnóstico Diferencial , Encefalitis/diagnóstico , Femenino , Fiebre/diagnóstico , Glucocorticoides/administración & dosificación , Glucocorticoides/uso terapéutico , Enfermedad de Hashimoto/diagnóstico , Humanos , Inmunoterapia , Metilprednisolona/administración & dosificación , Metilprednisolona/uso terapéutico , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/etiología , Neoplasias Ováricas/cirugía , Intercambio Plasmático , Receptores de N-Metil-D-Aspartato/inmunología , Convulsiones/diagnóstico , Teratoma/tratamiento farmacológico , Teratoma/etiología , Teratoma/cirugía , Resultado del TratamientoRESUMEN
Struma ovarii is a monodermal variant of ovarian teratoma. Thyroid-type carcinoma arising in struma ovarii is rare. The most common type is papillary carcinoma, followed by typical follicular carcinoma. A 75-year-old hypertensive patient consulted for the sensation of a painless pelvic mass that has been progressing for six months. The abdominopelvic ultrasound showed a right lateralized abdominopelvic mass measuring 14x13x8 cm with a solid and cystic double component. The patient underwent a unilateral right adnexectomy. Grossly, the tumor was encapsulated and lobulated. On cut sections, it was solid brown whitish in color and gelatinous. On histological examination, it was formed of follicular structures of variable size filled with a dense colloid. From this goiter a malignant tumor proliferation arose, arranged in sheets, trabeculae and follicular structures, and the tumor cells were cubic or polyhedral moderately atypical with few mitotic figures. There were no papillary-like nuclear features. There was focal capsular and vascular invasion. Immunohistochemical study showed positive immunostaining of tumor cells with TTF1. Postoperative course was uneventful. The exact prognosis of thyroid-type carcinoma arising in struma ovarii is still unclear because of its rarity, inadequate follow-up, and the absence of consensus in diagnosis and treatment.
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Neoplasias Ováricas , Estruma Ovárico/complicaciones , Neoplasias de la Tiroides , Anciano , Carcinoma Papilar/diagnóstico , Carcinoma Papilar/etiología , Carcinoma Papilar/patología , Proteínas de Unión al ADN/análisis , Femenino , Humanos , Inmunohistoquímica , Neoplasias Ováricas/diagnóstico , Neoplasias Ováricas/etiología , Neoplasias Ováricas/patología , Neoplasias Ováricas/cirugía , Estruma Ovárico/diagnóstico , Estruma Ovárico/patología , Estruma Ovárico/cirugía , Teratoma/diagnóstico , Teratoma/etiología , Teratoma/patología , Neoplasias de la Tiroides/diagnóstico , Neoplasias de la Tiroides/etiología , Neoplasias de la Tiroides/patología , Factores de Transcripción/análisisRESUMEN
The safe utilization of induced pluripotent stem cell (iPSC) derivatives in clinical use is attributed to the complete elimination of the risk of forming teratomas after transplantation. The extent by which such a risk exists in immune-competent hosts is mostly unknown. Here, using humanized mice reconstituted with fetal hematopoietic stem cells and autologous thymus tissue (bone-liver-thymus humanized mice [Hu-BLT]) or following the adoptive transfer of peripheral blood mononuclear cells(PBMCs) (Hu-AT), we evaluated the capacity of immune cells to prevent or eliminate teratomas derived from human iPSCs (hiPSCs). Our results showed that the injection of hiPSCs failed to form teratomas in Hu-AT mice reconstituted with allogeneic or autologous PBMCs or purified natural killer (NK) cells alone. However, teratomas were observed in Hu-AT mice reconstituted with autologous PBMCs depleted from NK cells. In line with these results, Hu-BLT, which do not have functional NK cells, could not prevent the growth of teratomas. Finally, we found that established teratomas were not targeted by NK cells and instead were efficiently rejected by allogeneic but not autologous T cells in Hu-AT mice. Overall, our findings suggest that autologous hiPSC-derived therapies are unlikely to form teratomas in the presence of NK cells.
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Células Asesinas Naturales/inmunología , Células Madre Pluripotentes/inmunología , Teratoma/prevención & control , Traslado Adoptivo/efectos adversos , Adulto , Animales , Humanos , Leucocitos Mononucleares/trasplante , Ratones , Ratones Endogámicos NOD , Ratones SCID , Linfocitos T/inmunología , Teratoma/etiología , Teratoma/inmunología , Trasplante HeterólogoRESUMEN
Human pluripotent stem cells (hPSCs) are an essential cell source in tissue engineering, studies of development, and disease modeling. Efficient, broadly amenable protocols for rapid lineage induction of hPSCs are of great interest in the stem cell biology field. We describe a simple, robust method for differentiation of hPSCs into mesendoderm in defined conditions utilizing single-cell seeding (SCS) and BMP4 and Activin A (BA) treatment. BA treatment was readily incorporated into existing protocols for chondrogenic and endothelial progenitor cell differentiation, while fine-tuning of BA conditions facilitated definitive endoderm commitment. After prolonged differentiation in vitro or in vivo, BA pretreatment resulted in higher mesoderm and endoderm levels at the expense of ectoderm formation. These data demonstrate that SCS with BA treatment is a powerful method for induction of mesendoderm that can be adapted for use in mesoderm and endoderm differentiation.
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Diferenciación Celular/genética , Mesodermo/citología , Mesodermo/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Transcripción Genética , Activinas/farmacología , Proteína Morfogenética Ósea 4/farmacología , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Endodermo/citología , Endodermo/metabolismo , Perfilación de la Expresión Génica , Humanos , Células Madre Pluripotentes/efectos de los fármacos , Análisis de la Célula Individual , Teratoma/etiología , Factores de Tiempo , TranscriptomaRESUMEN
Pluripotent stem cell (PSC) differentiation in vitro represents a powerful and tractable model to study mammalian development and an unlimited source of cells for regenerative medicine. Within hematology, in vitro PSC hematopoiesis affords novel insights into blood formation and represents an exciting potential approach to generate hematopoietic and immune cell types for transplantation and transfusion. Most studies to date have focused on in vitro hematopoiesis from mouse PSCs and human PSCs. However, differences in mouse and human PSC culture protocols have complicated the translation of discoveries between these systems. We recently developed a novel chemical media formulation, expanded potential stem cell medium (EPSCM), that maintains mouse PSCs in a unique cellular state and extraembryonic differentiation capacity. Herein, we describe how EPSCM can be directly used to stably maintain human PSCs. We further demonstrate that human PSCs maintained in EPSCM can spontaneously form embryoid bodies and undergo in vitro hematopoiesis using a simple differentiation protocol, similar to mouse PSC differentiation. EPSCM-maintained human PSCs generated at least two hematopoietic cell populations, which displayed distinct transcriptional profiles by RNA-sequencing (RNA-seq) analysis. EPSCM also supports gene targeting using homologous recombination, affording generation of an SPI1 (PU.1) reporter PSC line to study and track in vitro hematopoiesis. EPSCM therefore provides a useful tool not only to study pluripotency but also hematopoietic cell specification and developmental-lineage commitment.
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Medios de Cultivo/farmacología , Hematopoyesis/efectos de los fármacos , Células Madre Embrionarias Humanas/efectos de los fármacos , Células Madre Pluripotentes/efectos de los fármacos , Animales , Técnicas de Cultivo de Célula/métodos , Ciclo Celular , Linaje de la Célula , Células Cultivadas , Técnicas de Reprogramación Celular , Cuerpos Embrioides/efectos de los fármacos , Fibroblastos/citología , Genes Reporteros , Células Madre Embrionarias Humanas/citología , Humanos , Ratones , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de los fármacos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/trasplante , Análisis de Secuencia de ARN , Especificidad de la Especie , Trasplante de Células Madre/efectos adversos , Teratoma/etiologíaRESUMEN
Ethical and safety issues have rendered mesenchymal stem cells (MSCs) popular candidates in regenerative medicine, but their therapeutic capacity is lower than that of induced pluripotent stem cells (iPSCs). This study compared original, dental tissue-derived MSCs with re-differentiated MSCs from iPSCs (iPS-MSCs). CD marker expression in iPS-MSCs was similar to original MSCs. iPS-MSCs expressed higher in pluripotent genes, but lower levels in mesodermal genes than MSCs. In addition, iPS-MSCs did not form teratomas. All iPSCs carried mtDNA mutations; some shared with original MSCs and others not previously detected therein. Shared mutations were synonymous, while novel mutations were non-synonymous or located on RNA-encoding genes. iPS-MSCs also harbored mtDNA mutations transmitted from iPSCs. Selected iPS-MSCs displayed lower mitochondrial respiration than original MSCs. In conclusion, screening for mtDNA mutations in iPSC lines for iPS-MSCs can identify mutation-free cell lines for therapeutic applications. [BMB Reports 2019; 52(12): 689-694].
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ADN Mitocondrial/genética , Genoma Mitocondrial , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Mitocondrias/genética , Animales , Diferenciación Celular , Línea Celular , Humanos , Células Madre Pluripotentes Inducidas/citología , Masculino , Células Madre Mesenquimatosas/citología , Ratones , Ratones SCID , Mitocondrias/metabolismo , Tercer Molar/citología , Tercer Molar/crecimiento & desarrollo , Tercer Molar/metabolismo , Mutación , Medicina Regenerativa , Teratoma/etiologíaRESUMEN
Mucopolysaccharidosis Type II (MPS II), also known as Hunter syndrome, is a rare X-linked genetic disease caused by mutations in the IDS gene encoding iduronate 2-sulfatase (I2S). This is a multisystem disorder with significant variation in symptoms. Here, we document a human induced pluripotent stem cell (iPSC) line generated from dermal fibroblasts of a patient with Hunter syndrome containing a hemizygous mutation of a 1â¯bp insertion at nucleotide 208 in exon 2 of the IDS gene. The generation of this line will allow development of cell-based models for drug development, as well as the study of disease pathophysiology.
Asunto(s)
Diferenciación Celular , Reprogramación Celular , Fibroblastos/patología , Glicoproteínas/genética , Células Madre Pluripotentes Inducidas/patología , Mucopolisacaridosis II/genética , Mutación , Teratoma/etiología , Animales , Células Cultivadas , Preescolar , Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mucopolisacaridosis II/patología , Fenotipo , Teratoma/patologíaRESUMEN
A recessive mutation in PLA2G6, which is known to cause a heterogeneous neurodegenerative clinical spectrum, has recently been shown to be responsible for autosomal-recessive familial forms of Parkinson's disease (PD). Here, we generated induced pluripotent stem cells (iPSCs) from the peripheral blood mononuclear cells of a female patient with a homozygous PLA2G6 c.991Gâ¯>â¯T (p.D331Y) mutation by using the Sendai-virus delivery system. The resulting iPSCs showed pluripotency confirmed by immunofluorescent staining for pluripotency markers and differentiated into the 3 germ layers in vivo. This cellular model will provide a good resource for further pathophysiological studies of PD.
Asunto(s)
Diferenciación Celular , Reprogramación Celular , Fosfolipasas A2 Grupo VI/genética , Células Madre Pluripotentes Inducidas/patología , Mutación , Enfermedad de Parkinson/genética , Teratoma/etiología , Adulto , Edad de Inicio , Animales , Células Cultivadas , Femenino , Homocigoto , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Leucocitos Mononucleares/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Enfermedad de Parkinson/patología , Fenotipo , Teratoma/patologíaRESUMEN
Mucopolysaccharidosis type III B (MPS IIIB) is a lysosomal storage disorder caused by mutations in the NAGLU gene encoding N-acetylglucosaminidase. Here, we report the generation of a human induced pluripotent stem cell (iPSC) line from dermal fibroblasts of a MPS IIIB patient. The iPSC line has homozygous mutations of G>A transversion at nucleotide 457 of the NAGLU gene (457G>A), resulting in the substitution of lysine for glutamic acid at codon 153 (Glu153Lys). This iPSC line allows for the study of disease phenotypes and pathophysiology as well as disease modeling in human cells.
Asunto(s)
Acetilglucosaminidasa/genética , Homocigoto , Células Madre Pluripotentes Inducidas/patología , Mucopolisacaridosis III/genética , Mucopolisacaridosis III/patología , Mutación , Teratoma/etiología , Animales , Células Cultivadas , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Lactante , Ratones , Ratones Endogámicos NOD , Ratones SCID , Fenotipo , Teratoma/patologíaRESUMEN
ADNP syndrome is an intellectual disability associated with Autism spectrum disorder caused by mutations in ADNP. We generated an iPSC line from an ADNP syndrome pediatric patient harboring the mutation p.Trp719* (GENYOi004-A). Peripheral blood mononuclear cells were reprogrammed using a non-transmissible form of Sendai viruses expressing the four Yamanaka factors (Oct3/4, SOX2, KLF4 and c-MYC). Characterization of GENYOi004-A included mutation analysis of ADNP by allele-specific PCR, genetic identity by Short Tandem Repeats polymorphism profiling, alkaline phosphatase enzymatic activity, expression of pluripotency-associated factors and pluripotency studies in vivo. GENYOi004-A will be useful to evaluate ADNP syndrome alterations at early developmental stages.