Generating mature lineages from induced pluripotent stem cells with MECP2 disruption
Inventors
Rajesh, Deepika • Strouse, Anne • BURTON, Sarah • MUNN, Christie • Swanson, Bradley
Assignees
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Publication Number
US-10961505-B2
Publication Date
2021-03-30
Expiration Date
Abstract
Provided herein are methods for the efficient in vitro maintenance, expansion, culture, and/or differentiation of pluripotent cells with disruption of the MeCP2 gene into various erythroid, myeloid, lymphoid, or endoderm lineages, particularly mature erythrocytes. The pluripotent cells may be maintained and differentiated under defined conditions; thus, the use of mouse feeder cells or serum is not required in certain embodiments for the differentiation of the precursor cells.
Core Innovation
The invention relates to differentiating hematopoietic precursor cells (HPCs) derived from pluripotent stem cells (PSCs) into erythroid, lymphoid or myeloid cells by culturing HPCs that comprise disrupted Methyl-CpG Binding Protein 2 (MeCP2), and differentiating the HPCs to produce erythroid, myeloid or lymphoid cells. Disruption of MeCP2 is positioned as the central enabling feature for the differentiation outcomes.
The disclosed embodiments include HPCs that comprise non-functional MeCP2 with essentially no binding to methylated DNA and HPCs in which MeCP2 disruption occurs through loss by truncation, exon skipping or mutation, such that MeCP2 disrupted activity/function is achieved in the lineage differentiation process. The document further describes differentiation approaches in which the MeCP2-disrupted PSC or HPC intermediates are cultured through defined media and conditions to generate lineage-restricted progenitors and mature cells.
Example outcomes described include HPC production and erythroid differentiation with increased adult beta-globin and enucleation, lymphoid differentiation to T cells, NK cells and NK/T cells under specified hypoxia and culture formats, and myeloid differentiation including microglia enrichment with marker-defined identity. The invention also describes differentiation of definitive endoderm (DE) cells comprising disrupted MeCP2 into endodermal cells.
Claims Coverage
Two independent claims are provided. The inventive coverage centers on differentiating lineage outputs from PSC-derived intermediates by incorporating disrupted MeCP2 into HPCs, and separately into definitive endoderm (DE) cells prior to downstream differentiation into endodermal cells.
Differentiating MeCP2-disrupted HPCs into blood lineages
A method of differentiating hematopoietic precursor cells (HPCs) derived from pluripotent stem cells (PSCs) into erythroid, lymphoid or myeloid cells comprising culturing HPCs that comprise disrupted Methyl-CpG Binding Protein 2 (MeCP2), and differentiating the HPCs to produce erythroid, myeloid or lymphoid cells.
Differentiating MeCP2-disrupted definitive endoderm into endodermal cells
A method of differentiating definitive endoderm (DE) cells derived from PSCs into endodermal cells comprising obtaining DE cells that comprise disrupted Methyl-CpG Binding Protein 2 (MeCP2) and differentiating the DEs to produce endodermal cells.
Overall, the claim set covers MeCP2 disruption in PSC-derived intermediates as a preparatory requirement before differentiating into specified mature lineages, with one independent claim directed to HPC-derived erythroid/lymphoid/myeloid cells and another directed to DE-derived endodermal cells.
Stated Advantages
Enhances erythroid maturation, including earlier/higher CD235 expression, increased β-globin and enucleation.
Improves emergence of erythroid and HPC markers.
Increases T/NK differentiation outcomes, including CD3+/CD8+ T cells and NK/T cells derived from MeCP2KO HPCs.
Increases microglia purity (marker-positive populations after HPC-to-microglia differentiation).
Supports differentiation into additional lineages including definitive endoderm/hepatocytes with improved maturation markers such as albumin.
Documented Applications
Differentiation of PSC-derived HPCs into erythroid cells, including erythroid maturation outcomes such as CD235 expression, β-globin increase, and enucleation.
Differentiation of PSC-derived HPCs into lymphoid cells, including T cells/NK/T cell outcomes under hypoxia with Retronectin-DLL4.
Differentiation of PSC-derived HPCs into microglia, with increased microglia purity after HPC-to-microglia differentiation.
Differentiation into definitive endoderm/hepatocytes from PSC-derived lineages, with improved hepatocyte maturation markers such as albumin.
Differentiation of definitive endoderm (DE) cells derived from PSCs into endodermal cells, including downstream production of beta cells and more mature hepatocytes.
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