RESUMEN
Human ESC and iPSC are an attractive source of cells of high quantity and purity to be used to elucidate early human development processes, for drug discovery, and in clinical cell therapy applications. To efficiently differentiate pluripotent cells into a pure population of hematopoietic progenitors we have developed a new 2-dimensional, defined and highly efficient protocol that avoids the use of feeder cells, serum or embryoid body formation. Here we showed that a single matrix protein in combination with growth factors and a hypoxic environment is sufficient to generate from pluripotent cells hematopoietic progenitors capable of differentiating further in mature cell types of different lineages of the blood system. We tested the differentiation method using hESCs and 9 iPSC lines generated from different tissues. These data indicate the robustness of the protocol providing a valuable tool for the generation of clinical-grade hematopoietic cells from pluripotent cells.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/citología , Células Madre Hematopoyéticas/citología , Células Madre Pluripotentes/citología , Medio de Cultivo Libre de Suero , Citometría de Flujo , Humanos , Técnicas In Vitro , Oxígeno/metabolismoRESUMEN
OBJECTIVE: Human embryonic stem (ES) cells provide a unique model for studying the development and function of human tissues and have proven utility in a number of areas. However, results from ES cell-based studies have been limited by the paucity of information available about early human hematopoietic development. METHODS: To better understand early development of the hematopoietic lineage, we use microarray analysis to examine the temporal patterns of gene expression in embryoid bodies derived from human ES cells, focusing around the time of the emergence of definitive hematopoiesis. We use an empirical Bayes hierarchical modeling approach, called EBarrays, to classify genes into each of the possible temporal patterns of gene expression for five different time points, and correlate those patterns with the emergence of hematopoiesis. RESULTS: We find a distinct group of genes previously identified as important in adult hematopoietic self-renewal (such as PIK3R1, ABCB1/MDR-1, RGS18, IRS1, SENP6/SUMO-1, and Wnt5A, etc.) temporally correlates with the emergence of the definitive hematopoiesis. Microarray-based results are further supported via flow cytometry and reverse transcription-polymerase chain reaction studies. CONCLUSION: The novel genes demonstrating the same expression pattern as this group could further facilitate the understanding of the molecular mechanisms of embryonic hematopoiesis.
Asunto(s)
Diferenciación Celular/genética , Células Madre Embrionarias/fisiología , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis/genética , Análisis por Micromatrices , Antígenos CD34/metabolismo , Secuencia de Bases , Línea Celular , Linaje de la Célula , Células Cultivadas , Células Madre Embrionarias/química , Humanos , Datos de Secuencia Molecular , Regulación hacia ArribaRESUMEN
We have previously reported that high concentrations of basic fibroblast growth factor (bFGF) support feeder-independent growth of human embryonic stem (ES) cells, but those conditions included poorly defined serum and matrix components. Here we report feeder-independent human ES cell culture that includes protein components solely derived from recombinant sources or purified from human material. We describe the derivation of two new human ES cell lines in these defined culture conditions.