Multi-lineage hematopoietic precursor cell production by genetic programming
Inventors
Yu, Junying • Vodyanyk, Maksym A. • Sasaki, Jeffrey • Rajesh, Deepika • BURTON, Sarah A.
Assignees
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Publication Number
US-10865381-B2
Publication Date
2020-12-15
Expiration Date
Abstract
The present disclosure generally regards methods and compositions for providing multi-lineage hematopoietic precursor cells from pluripotent stem cells (PSCs). The PSCs comprise an expression construct encoding an ETS/ERG gene, GATA2 and HOXA9. Also provided are methods for providing hematopoietic stem cells capable of long-term engraftment in mammals, such as humans. Further provided are therapeutic compositions including the provided hematopoietic stem cells and precursors of hematopoietic cells, and methods of using such for the treatment of subjects.
Core Innovation
The disclosure describes an in vitro method for producing hematopoietic precursor cells from pluripotent stem cells. The method provides pluripotent stem cells comprising at least one expression construct encoding hematopoietic precursor programming genes including ETS/ERG, GATA2, and HOXA9, wherein the genes are co-expressed. The pluripotent stem cells are induced to express the hematopoietic precursor programming genes to produce hematopoietic precursor cells.
The method further includes culturing the hematopoietic precursor cells in which expression of the hematopoietic precursor programming genes is no longer induced, to produce a population comprising at least 70% multi-lineage hematopoietic precursor cells with expression of CD34 and CD43. The disclosure characterizes producing multi-lineage hematopoietic precursor cells and identifies marker panels that include CD34, CD43, CD45, CD33, CD235a, and CD41a.
The disclosure also describes related constructs and expression control that can be used to program pluripotent stem cells, including expression constructs encoding ERG, GATA2, and HOXA9 under the control of a single promoter, and selectable or inducible expression systems. It further describes optionally adding additional hematopoietic stem cell programming genes to produce hematopoietic stem cells with long-term engraftment.
Claims Coverage
The document contains two independent claims. Across these independent claims, the coverage is centered on programming pluripotent stem cells with ETS/ERG, GATA2, and HOXA9, then culturing to obtain at least 70% multi-lineage hematopoietic precursor cells expressing CD34 and CD43, with subsequent loss of continued expression or coexpression of the programming genes.
In vitro production of multi-lineage HPCs via induced co-expression of ETS/ERG, GATA2, HOXA9
An in vitro method for producing hematopoietic precursor cells from pluripotent stem cells comprising providing pluripotent stem cells comprising at least one expression construct encoding hematopoietic precursor programming genes comprising ETS/ERG, GATA2, and HOXA9 genes co-expressed; culturing the PSCs wherein the PSCs are induced to express the hematopoietic precursor programming genes, thereby producing hematopoietic precursor cells; and further culturing the HPCs wherein expression of the hematopoietic precursor programming genes is no longer induced to produce a population of cells comprising at least 70% multi-lineage HPCs with expression of CD34 and CD43.
In vitro production of multi-lineage HPCs via single-promoter ERG, GATA2, HOXA9 construct followed by cessation of coexpression
An in vitro method for producing hematopoietic precursor cells from pluripotent stem cells comprising providing pluripotent stem cells comprising an expression construct encoding ERG, GATA2, and HOXA9 under the control of a single promoter; culturing the pluripotent stem cells with coexpression of ERG, GATA2, and HOXA9, thereby producing hematopoietic precursor cells; and further culturing the HPCs without coexpression of ERG, GATA2, and HOXA9 to produce a population of cells comprising at least 70% multi-lineage HPCs with expression of CD34 and CD43.
Overall, the independent claims require programming PSCs with ETS/ERG, GATA2, and HOXA9 to generate hematopoietic precursor cells, followed by culturing conditions that stop induced expression or coexpression of the programming genes, resulting in a population comprising at least 70% multi-lineage hematopoietic precursor cells expressing CD34 and CD43.
Stated Advantages
Produces a population comprising at least 70% multi-lineage hematopoietic precursor cells with expression of CD34 and CD43.
Enables in vitro generation of hematopoietic precursor cells from pluripotent stem cells using ETS/ERG, GATA2, and HOXA9 programming genes.
Documented Applications
In vitro production of hematopoietic precursor cells from pluripotent stem cells, including producing a multi-lineage HPC population expressing CD34 and CD43.
Optionally producing hematopoietic stem cells with long-term engraftment.
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