RESUMEN
Pluripotency is characterized by specific transcription factors such as OCT4, NANOG, and SOX2, but also by pluripotency-associated microRNAs (miRs). Somatic cells can be reprogrammed by forced expression of these factors leading to induced pluripotent stem cells (iPSCs) with characteristics similar to embryonic stem cells (ESCs). However, current reprogramming strategies are commonly based on viral delivery of the pluripotency-associated factors, which affects the integrity of the genome and impedes the use of such cells in any clinical application. In an effort to establish nonviral, nonintegrating reprogramming strategies, we examined the influence of hypoxia on the expression of pluripotency-associated factors and the ESC-specific miR-302 cluster in primary and immortalized mesenchymal stromal cells (MSCs). The combination of hypoxia and fibroblast growth factor 2 (FGF2) treatments led to the induction of OCT4 and NANOG in an immortalized cell line L87 and primary MSCs, accompanied with increased doubling rates and decreased senescence. Most importantly, the endogenous ECS-specific cluster miR-302 was induced upon hypoxic culture and FGF2 supplementation. Hypoxia also improved reprogramming of MSCs via episomal expression of pluripotency factors. Thus, our data illustrate that hypoxia in combination with FGF2 supplementation efficiently facilitates reprogramming of MSCs.
Asunto(s)
Desdiferenciación Celular , Células Madre Embrionarias/metabolismo , Células Madre Mesenquimatosas/metabolismo , MicroARNs/biosíntesis , Familia de Multigenes , Células Madre Pluripotentes/metabolismo , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Transformada , Células Madre Embrionarias/citología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Proteínas de Homeodominio/metabolismo , Humanos , Células Madre Mesenquimatosas/citología , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/citologíaRESUMEN
BACKGROUND: Various techniques have been employed to detect BCR-ABL kinase domain mutations in patients with chronic myeloid leukemia who are resistant to imatinib. This has led to different reported frequencies of mutations and the finding of a heterogeneous pattern of individual mutations. DESIGN AND METHODS: We compared direct sequencing alone and in combination with denaturing high-performance liquid chromatography and two high-sensitivity allele-specific oligonucleotide polymerase chain reaction approaches for analysis of BCR-ABL mutations in 200 blinded cDNA samples prior to and during second-line dasatinib or nilotinib therapy in patients with chronic myeloid leukemia in whom imatinib treatment had failed. RESULTS: One hundred and fourteen mutations were detected by both direct sequencing alone or in combination with high performance liquid chromatography and 13 mutations were additionally detected by the combined technique. Eighty of 83 mutations (96%) within a selected panel of 11 key mutations were confirmed by both allele-specific oligonucleotide polymerase chain reaction techniques and 62 mutations were identified in addition to those detected by combined liquid chromatography and direct sequencing, indicating the presence and a high prevalence of low-level mutations in this cohort of patients. Furthermore, 125 mutations were detected by only one allele-specific oligonucleotide polymerase chain reaction technique. Pre-existing mutations were traceable 4.5 months longer and emerging clones were detectable 3.0 months earlier by allele-specific oligonucleotide polymerase chain reaction than by direct sequencing together with liquid chromatography. CONCLUSIONS: Our results suggest that denaturing high performance liquid chromatography combined with direct sequencing is a reliable screening technique for the detection of BCR-ABL kinase domain mutations. Allele-specific oligonucleotide polymerase chain reaction further increases the number of detected mutations and indicates a high prevalence of mutations at a low level. The clinical impact of such low-level mutations remains uncertain and requires further investigation. Allele-specific oligonucleotide polymerase chain reaction allows detection of defined mutations at a lower level than does denaturing high performance liquid chromatography combined with direct sequencing and may, therefore, provide clinical benefit by permitting early reconsideration of therapeutic strategies.
Asunto(s)
Resistencia a Antineoplásicos/genética , Proteínas de Fusión bcr-abl/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Mutación , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteínas Tirosina Quinasas/genética , Pirimidinas/administración & dosificación , Tiazoles/administración & dosificación , Adulto , Anciano , Benzamidas , Análisis Mutacional de ADN , Dasatinib , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Masculino , Persona de Mediana Edad , Piperazinas/administración & dosificación , Reacción en Cadena de la Polimerasa , Estructura Terciaria de Proteína/genética , Proteínas Tirosina Quinasas/antagonistas & inhibidoresRESUMEN
OBJECTIVE: The ABCG2 transporter has been identified as a determinant of the side population (SP) in murine bone marrow and a potential marker for primitive stem cells. To assess the potential of the SP phenotype and ABCG2 expression to identify stem cells in human umbilical cord blood (hUCB), we have examined directly the relationship between SP; expression of ABCG2, CD133, and CD34; and hematopoietic potential in UCB samples. MATERIALS AND METHODS: Multicolor fluorescence activated cell sorting analysis was combined with the Hoechst SP procedure to allow the simultaneous detection of the SP phenotype together with surface markers in cells from fresh and cryopreserved UCB. Sorted populations were analyzed for cobblestone area-forming cell (CAFC) activity and by quantitative reverse transcriptase polymerase chain reaction for expression of mRNA from the ABC transporters ABCG2, MDR1, and MRP1. ABCG2(+) cells were enriched by magnetic-activated cell sorting for stringent analysis. RESULTS: hUCB-derived SP cells were negative for ABCG2, but comprise approximately 20% CD133(+)/CD34(+) cells. Sorted SP cells from UCB were enriched 20-fold for week 13 CAFC activity, while magnetic-activated cell sorting-enriched ABCG2(+) cells retained no hematopoietic activity either in CAFC or liquid cultures. There was no significant difference in the SP frequency, immunophenotype, or CAFC potential of fresh and cryopreserved UCB. ABCG2 mRNA was not enriched in the SP and was specifically diminished ninefold in CD133 cells, which were eightfold enriched for MDR1 mRNA. CONCLUSION: We find no evidence for an association of ABCG2 with SP activity or hematopoietic progenitor function in hUCB.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/biosíntesis , Antígenos de Diferenciación/biosíntesis , Sangre Fetal/metabolismo , Regulación de la Expresión Génica/fisiología , Células Madre Hematopoyéticas/metabolismo , Proteínas de Neoplasias/biosíntesis , Antígeno AC133 , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Animales , Antígenos CD/biosíntesis , Antígenos CD34/biosíntesis , Sangre Fetal/citología , Glicoproteínas/biosíntesis , Células Madre Hematopoyéticas/citología , Humanos , Ratones , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/biosíntesis , PéptidosRESUMEN
UNLABELLED: In recent years, a large number of groups studied the fate of human stem cells in livers of immunodeficient animals. However, the interpretation of the results is quite controversial. We transplanted 4 different types of human extrahepatic precursor cells (derived from cord blood, monocytes, bone marrow, and pancreas) into livers of nonobese diabetic/severe combined immunodeficiency mice. Human hepatocytes were used as positive controls. Tracking of the transplanted human cells could be achieved by in situ hybridization with alu probes. Cells with alu-positive nuclei stained positive for human albumin and glycogen. Both markers were negative before transplantation. However, cells with alu-positive nuclei did not show a hepatocyte-like morphology and did not express cytochrome P450 3A4, and this suggests that these cells represent a mixed cell type possibly resulting from partial transdifferentiation. Using antibodies specific for human albumin, we also observed a second human albumin-positive cell type that could be clearly distinguished from the previously described cells by its hepatocyte-like morphology. Surprisingly, these cells had a mouse and not a human nucleus which is explained by transdifferentiation of human cells. Although it has not yet been formally proven, we suggest horizontal gene transfer as a likely mechanism, especially because we observed small fragments of human nuclei in mouse cells that originated from deteriorating transplanted cells. Qualitatively similar results were obtained with all 4 human precursor cell types through different routes of administration with and without the induction of liver damage. CONCLUSION: We observed evidence not for transdifferentiation but instead for a complex situation including partial differentiation and possibly horizontal gene transfer.