Serum- and Stromal Cell-Free Hypoxic Generation of Embryonic Stem Cell-Derived Hematopoietic Cells In Vitro, Capable of Multilineage Repopulation of Immunocompetent Mice

Abstract

Induced pluripotent stem cells (iPSCs) may become a promising source for the generation of patientspecific hematopoietic stem cells (HSCs) in vitro. A crucial prerequisite will be the availability of reliable protocols for the directed and efficient differentiation toward HSCs. So far, the most robust strategy for generating HSCs from pluripotent cells in vitro has been established in the mouse model involving ectopic expression of the human transcription factor HOXB4. However, most differentiation protocols include coculture on a xenogenic stroma cell line and the use of animal serum. Involvement of any of both would pose a major barrier to the translation of those protocols to human autologous iPSCs intended for clinical use. Therefore, we asked whether long-term repopulating HSCs can, in principle, be generated from embryonic stem cells without stroma cells or serum. Here, we showed that long-term multilineage engraftment could be accomplished in immunocompetent mice when HSCs were generated in serum-free medium without stroma cell support and when hypoxic conditions were used. Under those conditions, HOXB4؉ embryonic stem cell-derived hematopoietic stem and progenitor cells were immunophenotypically similar to definitive bone marrow resident E-SLAM ؉ (CD150 ؉ CD48 − CD45 ؉ CD201 ؉ ) HSCs. Thus, our findings may ease the development of definitive, adult-type HSCs from pluripotent stem cells, entirely in vitro. STEM CELLS TRANSLATIONAL MEDICINE 2012;1:581-591

Generation of iPSCs from Genetically Corrected Brca2 Hypomorphic Cells: Implications in Cell Reprogramming and Stem Cell Therapy

“…Neither the use of AM20.1B4 stromal cells nor the differentiation method based on the transduction of iPSC with the Lhx2 vector , improved the differentiation capacity of these cells (not shown). Moreover, although transplantations of mESC‐derived hematopoietic cells into irradiated recipients resulted in significant hematopoietic engraftments , no reproducible engraftments were observed when A16‐Ex7 c Brca2 Δ 27/ Δ 27 iPSCs were transplanted. In these transplants a reduced number (<1%) of Hox‐B4/EGFP + cells, which did not express hematopoietic markers, could be observed for up to 30 days post‐transplantation in peripheral blood (PB).…”

Low oxygen tension enhances the generation of lung progenitor cells from mouse embryonic and induced pluripotent stem cells

“…), hematopoietic (Lesinski et al. ), and chondrogenic (Koay and Athanasiou ; Adesida et al. ) cells among others.…”

“…), and hematopoietic cells (Lesinski et al. ). Recently, differentiation of human ESC into the oligodendrocyte lineage has been accomplished under physiologically relevant 3% oxygen conditions (Stacpoole et al.…”

HOXB4 Increases Runx1 Expression to Promote the de novo Formation of Multipotent Hematopoietic Cells

“…These cells further matured towards CD41-CD45+ cells, an important criterion for definitive, adult HSCs. In previous work, we and others have repeatedly shown that such ES cell-derived hematopoietic cells mediate multilineage, longterm repopulation after in vivo transplantation in mice [2,3,[5][6][7].…”

“…HOXB4 belongs to the family of homeotic selector proteins encoded by homeobox (HOX) genes which are well known for their importance to confer positional information to cells and tissues during embryonic development and to control stem cell self-renewal and differentiation in the adult organism [4]. Human HOXB4, in particular, has repeatedly been demonstrated to enhance the hematopoietic potential of differentiating embryonic stem cells (ESCs) in vitro [1,3,[5][6][7][8][9] and to mediate an expansion of adult HSCs of mice and humans [10][11][12]. However, the underlying mechanisms how HOXB4 mediates these valuable effects are still not understood very well, thus making it a challenging process to decipher the best conditions for translating the results from the mouse to the human system.…”