Hematopoietic development from human induced pluripotent stem cells

Abstract

A decade of research on human embryonic stem cells (ESC) has paved the way for the discovery of alternative approaches to generating pluripotent stem cells. Combinatorial overexpression of a limited number of proteins linked to pluripotency in ESC was recently found to reprogram differentiated somatic cells back to a pluripotent state, enabling the derivation of isogenic (patient-specific) pluripotent stem cell lines. Current research is focusing on improving reprogramming protocols (e.g., circumventing the use of retroviral technology and oncoproteins), and on methods for differentiation into transplantable tissues of interest. In mouse ESC, we have previously shown that the embryonic morphogens BMP4 and Wnt3a direct blood formation via activation of Cdx and Hox genes. Ectopic expression of Cdx4 and HoxB4 enables the generation of mouse ESC-derived hematopoietic stem cells (HSC) capable of multilineage reconstitution of lethally irradiated adult mice. Here, we explore hematopoietic development from human induced pluripotent stem (iPS) cells generated in our laboratory. Our data show robust differentiation of iPS cells to mesoderm and to blood lineages, as shown by generation of CD34(+)CD45(+) cells, hematopoietic colony activity, and gene expression data, and suggest conservation of blood patterning pathways between mouse and human hematopoietic development.

FIGURE 1. Generation of blood cells from adult skin fibroblasts

For direct reprogramming of somatic cells, primary fibroblasts (shown here at day 28 of culture) undergo retroviral infection with embryonic genes (e.g. OCT4, SOX2, KLF4, MYC) and are afterwards grown on mouse embryonic feeder cells (MEF) in ESC growth medium. After several weeks of culture, iPS colonies displaying ESC-like morphology appear (shown here: hFib2-iPS5 colonies). iPS cells can be maintained in an undifferentiated state as a cell line or differentiated in embryoid bodies (EB, shown here hFib2-iPS5 at day 10 of differentiation) to tissues of interest, e.g. blood (shown here, myeloid colony at day 10 after plating day 17 EB-derived cells into methylcellulose supplemented with hematopoietic cytokines). Pictures were taken at 40× magnification.

FIGURE 2. Blood development from iPS cells

(A) Day 17 EB-derived, differentiated hFib2-iPS5 cells show robust formation of CD34⁺ and CD45⁺ and CD34⁺CD45⁺ cells. (B–C) Hematopoietic colony formation upon plating in methylcellulose CFU assays. (B) Shown are colony numbers per 12.500 plated day 17 EB-derived cells and (C) pictures of different colony types (erythroid, macrophage, granulocyte-macrophage, granulocyte-erythroid-macrophage-megakaryocyte) taken at 100× magnification. (D) Time course of quantitative RT-PCR analysis of differentiating hFib2-iPS5 shows expression of mesodermal (BRACHYURY) and hematopoietic (SCL, GATA2) gene expression, as well as expression of embryonic homeobox genes previously implicated in murine blood development (CDX1, CD2, CDX4 and HOXA9). Numbers represent fold relative gene expression, as compared to the expression in undifferentiated iPS5 cells (day 0).

FIGURE 3. BMP4 promotes hematopoiesis from human iPS cells

(A) The BMP downstream signaling components SMAD1/5/8 show phosphorylation in iPS5 cells cultured in basic differentiation medium;addition of BMP4 enhances this signal as measured by intracellular flow cytometry; (B) Addition of BMP4 to cultures in basic differentiation medium enhances the formation of CD34⁺CD45⁺ cells and (C) upregulates mesodermal (BRACHYURY, as measured in day 6 EB) and hematopoietic genes (SCL, GATA2, as measured in day 9 EB).