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Targeted organ generation using Mixl1-inducible mouse pluripotent stem cells in blastocyst complementation.

ABSTRACT: Generation of functional organs from patients' own cells is one of the ultimate goals of regenerative medicine. As a novel approach to creation of organs from pluripotent stem cells (PSCs), we employed blastocyst complementation in organogenesis-disabled animals and successfully generated PSC-derived pancreas and kidneys. Blastocyst complementation, which exploits the capacity of PSCs to participate in forming chimeras, does not, however, exclude contribution of PSCs to the development of tissues-including neural cells or germ cells-other than those specifically targeted by disabling of organogenesis. This fact provokes ethical controversy if human PSCs are to be used. In this study, we demonstrated that forced expression of Mix-like protein 1 (encoded by Mixl1) can be used to guide contribution of mouse embryonic stem cells to endodermal organs after blastocyst injection. We then succeeded in applying this method to generate functional pancreas in pancreatogenesis-disabled Pdx1 knockout mice using a newly developed tetraploid-based organ-complementation method. These findings hold promise for targeted organ generation from patients' own PSCs in livestock animals.

SUBMITTER: Kobayashi T

PROVIDER: S-EPMC4291089 | biostudies-other | 2015 Jan

REPOSITORIES: biostudies-other

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Project description:Genetically modified mice are commonly generated by the microinjection of pluripotent embryonic stem (ES) cells into wild-type host blastocysts1, producing chimeric progeny that require breeding for germline transmission and homozygosity of modified alleles. As an alternative approach and to facilitate studies of the immune system, we previously developed RAG2-deficient blastocyst complementation2. Because RAG2-deficient mice cannot undergo V(D)J recombination, they do not develop B or T lineage cells beyond the progenitor stage2: injecting RAG2-sufficient donor ES cells into RAG2-deficient blastocysts generates somatic chimaeras in which all mature lymphocytes derive from donor ES cells. This enables analysis, in mature lymphocytes, of the functions of genes that are required more generally for mouse development3. Blastocyst complementation has been extended to pancreas organogenesis4, and used to generate several other tissues or organs5-10, but an equivalent approach for brain organogenesis has not yet been achieved. Here we describe neural blastocyst complementation (NBC), which can be used to study the development and function of specific forebrain regions. NBC involves targeted ablation, mediated by diphtheria toxin subunit A, of host-derived dorsal telencephalic progenitors during development. This ablation creates a vacant forebrain niche in host embryos that results in agenesis of the cerebral cortex and hippocampus. Injection of donor ES cells into blastocysts with forebrain-specific targeting of diphtheria toxin subunit A enables donor-derived dorsal telencephalic progenitors to populate the vacant niche in the host embryos, giving rise to neocortices and hippocampi that are morphologically and neurologically normal with respect to learning and memory formation. Moreover, doublecortin-deficient ES cells-generated via a CRISPR-Cas9 approach-produced NBC chimaeras that faithfully recapitulated the phenotype of conventional, germline doublecortin-deficient mice. We conclude that NBC is a rapid and efficient approach to generate complex mouse models for studying forebrain functions; this approach could more broadly facilitate organogenesis based on blastocyst complementation.

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Targeted organ generation using Mixl1-inducible mouse pluripotent stem cells in blastocyst complementation.

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