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Interspecies Chimerism with Mammalian Pluripotent Stem Cells  Wu et al., Cell  Volume 168, Issue 3, (January 2017) Journal Club February 16, 2017 …it remains unclear whether naive PSCs can be used to generate chimeras between more distantly related species.

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Steps in Embryogenesis naive state = unbiased developmental potential primed state = cells are poised for lineage differentiation

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Like naive rodent PSCs, naive hPSCs can potentially be used to generate interspecies chimeras for studying human development and disease, and producing functional human tissues via interspecies blastocyst complementation. Generating hPSC-derived interspecies chimeras, mainly in mouse, rather inefficient Interspecies chimera research of hPSCs using ungulates, e.g., pigs, cattle, and sheep, could lead to improved research models, as well as novel in vivo strategies for (1) generating human organs and tissues, (2) designing new drug screening methodologies, and (3) developing new human disease models Research using ungulates is currently lacking

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We first used rodent models to gain a better understanding of the factors and caveats underlying interspecies chimerism with PSCs. Labeled w/ humanized kusabira orange (hKO) surrogate mouse mothers Many of the chimeras developed into adulthood, and one chimera reached 2 years of age

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Chimera forming efficiencies of all rat PSC lines tested were ~ 20% Contribution of rat cells to a wide range of tissues and organs in both neonatal and aged rat-mouse chimeras We examined aging-related histone marks in both neonatal and aged chimeras and found that the 2-year-old chimera exhibited histone signatures characteristic of aging H3K9me3 - transcriptional repression H4K20me3 - silencing of repetitive DNA and transposons ?

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We quantified the degree of chimerism in different organs of the aged chimera via quantitative qPCR analysis of genomic DNA using a rat-specific primer the highest contribution observed in the heart (~10%) rats lack a gallbladder We observed the presence of gallbladders in rat-mouse chimeras Interestingly, rat cells contributed to the chimeric gallbladder and expressed the gallbladder epithelium marker EpCAM Mouse embryonic microenvironment was able to unlock a gallbladder developmental program in rat PSCs that is normally suppressed during rat development.

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Short intro: CRISPR technology MIT OriGene For proof-of-concept, we knocked out the Pdx1 gene in mouse by co-injecting Cas9 mRNA and Pdx1 single-guide RNA (sgRNA) into mouse zygotes. Pdx1 expression is restricted to the pancreas Pdx1-/- lack a pancreas and die within a few days after birth Rat PCSs complemented mouse Pdx1-/-

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PSC-derivatives were enriched in the neonatal pancreas of Pdx1 -/- mice and expressed alpha-AMYLAYSE, a pancreatic enzyme that helps digest carbohydrates Immunofluorescence image showing that some pancreatic endothelial cells, as marked by a CD31 antibody, were not derived from rat PSCs. Nkx2.5 plays a critical role in early stages of cardiogenesis. Mice lacking Nkx2.5 typically die around E10.5 Enrichment of rat cells in the heart of an E10.5 Nkx2.5 -/- mouse The embryo size was restored to normal Mice homozygous for a Pax6 loss-of-function mutation lack eyes Formation of chimeric eyes enriched with rat cells in Pax6 -/- mouse neonate

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PSC-derivatives were enriched in the neonatal pancreas of Pdx1 -/- mice and expressed alpha-AMYLAYSE, a pancreatic enzyme that helps digest carbohydrates Immunofluorescence image showing that some pancreatic endothelial cells, as marked by a CD31 antibody, were not derived from rat PSCs. Nkx2.5 plays a critical role in early stages of cardiogenesis. Mice lacking Nkx2.5 typically die around E10.5 Enrichment of rat cells in the heart of an E10.5 Nkx2.5 -/- mouse The embryo size was restored to normal Mice homozygous for a Pax6 loss-of-function mutation lack eyes Formation of chimeric eyes enriched with rat cells in Pax6 -/- mouse neonate In sum, for the pancreas, heart, and eye, we successfully generated chimerized organs that were enriched with rat cells.

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It has not yet been tested whether naive rodent PSCs can contribute to chimera formation when using a non-rodent host 2. Inject rat ESCs + mouse iPSCs into pig blastocysts followed by ET to recipient sows. Determine the chimeric contribution of rodent cells in pig embryos: detection of fluorescence (hKO) signal, immunohistochemical (IHC) labeling of embryo sections with an anti-hKO antibody, and genomic PCR with mouse- or rat-specific primers targeting mito- chondrial DNA (mtDNA) 1. Use rodent models to gain a better understanding of the factors and caveats underlying interspecies chimerism with PSCs.

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+ lack of any immunohistochemical signal (1) (2) (3) Taken together, although naive rodent PSCs can robustly contribute to rodent-specific interspecies chimeras, our results show that these cells are incapable of contributing to normal embryonic development in pigs.

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2. Inject rat ESCs + mouse iPSCs into pig blastocysts followed by ET to recipient sows. 1. Use rodent models to gain a better understanding of the factors and caveats underlying interspecies chimerism with PSCs. 3. Systematically evaluate the chimeric competency of hPSCs in ungulate embryos. We generated hiPSCs using several reported naive PSC culture methods All 4 lines could be stably maintained long term in culture, preserving normal karyotypes and the homogeneous, nuclear localization of OCT4 protein Oct4 protein - absolutely required for the stemness properties of murine and primate embryonic stem cells. Label hiPSCs with either GFP or hKO fluorescence markers to facilitate identification later on

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3A. Ability of hiPSCs to integrate into the inner cell mass (ICM) of a blastocyst

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3A. Ability of hiPSCs to integrate into the inner cell mass (ICM) of a blastocyst Number of hiPSCs that integrated into the cattle (left) and pig (right) ICMs after ten hiPSCs were injected into the blastocyst followed by 2 days of in vitro culture. Red line, the average number of ICM-incorporated hiPSCs. SOX2 - essential for maintaining pluripotency

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3B. Investigate if any of the hiPSCs, which showed robust ICM incorporation in preimplantation blastocysts, could contribute to post-implantation development following ET. 41 surrogate sows received 30–50 embryos each, resulting in 18 pregnancies Collection of embryos between day 21-28 of development resulted in the harvesting of 186 embryos: 43 from 2iLD-hiPSCs, 64 from FAC- hiPSCs, 39 from 4i-hiPSCs, and 40 from NHSM-hiPSCs

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Further analyses of the 186 embryos (99 normal size + 87 retarded) (1) fluorescence All normal size FO+ embryos derived from 2iLD-hiPSCs, 4i-hiPSCs, or NHSM-hiPSCs showed a very limited fluorescence signal

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In contrast, normal size FO+ FAC-hiPSC-derived embryos typically exhibited a more robust fluorescence signal FO+ IHC w/ antibodies detecting GFP or hKO For FAC-hiPSC-derived embryos, we confirmed via IHC analysis (using an anti-GFP antibody) that they contained more human cells Stained additional sections using antibodies against TUJ1, EPCAM, SMA, CK8, and HNF3b and observed differentiation of FAC-hiPSCs into different cell lineages. In addition, these cells were found negative for OCT4, a pluripotency marker (data not shown). Moreover, the presence of human cells was further veri- fied with a human-specific HuNu antibody staining

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PCR, using human specific Alu sequence primer

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Interspecies Chimerism with Mammalian Pluripotent Stem Cells  Wu et al., Cell  Volume 168, Issue 3, (January 2017) Journal Club February 16, 2017 …it remains unclear whether naive PSCs can be used to generate chimeras between more distantly related species. The levels of chimerism from all hiPSCs in pig embryos were much lower when compare to rat-mouse chimeras, which may reflect the larger evolutionary distance between human-pig than between rat-mouse.

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