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Cloning and Variation of Ground State Intestinal Stem Cells

ABSTRACT: Despite major advances with embryonic and induced pluripotent stem cells or lineage-committed, p63-expressing stem cells of stratified epithelia, we know less about the indigenous stem cells of the gastrointestinal tract, pancreas, liver, and other columnar epithelia which collectively resist cloning in their elemental states. Here we demonstrate the cloning of highly immature epithelial stem cells from defined regions of the human intestine and colon. We show that single cell-derived pedigrees can be propagated indefinitely while often sustaining minimal copy number and sequence variation. Despite prolonged cultivation, these pedigrees from disparate regions of the intestinal tract respond to identical differentiation signals by formation of epithelia with eponymous structural and gene expression features. These data suggest developmental patterning of cell-autonomous commitment programs in stem cells that enforce specialization along the gastrointestinal tract and predict the utility of these cells in disease modeling and regenerative medicine.

ORGANISM(S): Homo sapiens

PROVIDER: GSE57584 | GEO | 2015/05/29

SECONDARY ACCESSION(S): PRJNA246737

REPOSITORIES: GEO

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Project description:Interstitial cells of Cajal (ICC) are electrical pacemakers and mediators of neuromuscular neurotransmission in the gastrointestinal tract. Studies in organotypic cultures of intact gastric muscular wall tissues identified Kit(low)Cd44(+)Cd34(+)Insr(+)Igf1r(+) cells as local ICC progenitors (Lorincz et al., Gastroenterology 2008;134:1083-1093). Subsequently, conditionally immortalized Kit(low)Cd34(+) cells were isolated by immunomagnetic and fluorescent cell sorting and serial cloning from the gastric muscular wall of homozygous, 14-day-old H-2Kb-tsA58 mice and shown to share other phenotypic characteristics with in-situ ICC precursors (2xSCS70 cells; Bardsley et al., Gastroenterology 2010;139:942-952). 2xSCS70 cells were also found to express the ICC marker Ano1, be able to extensively self-renew in the absence of the SV40 tsA58-TAg when cultured in NeuroCultÂ® Neural Stem Cell Proliferation Medium (NSCPM; STEMCELL Technologies) and differentiate into Kit(+) ICC both in vitro and in vivo. Wild-type cells with Kit(low)Ano1(+)Cd44(+)Cd34(+)Insr(+)Igf1r(+) phenotype (2xSCS2F10 cells) were prospectively isolated from primary cultures of dissociated gastric muscular wall tissues from 7-day-old C57BL/6J mice by serial cloning following 150-day expansion in NSCPM containing 5% cell-free chick embryo extract (CEE; Accurate Chemical). Long-term culturing of both cell lines was associated with further decline in Kit expression while expression of Ano1 and other markers of ICC precursors was maintained. Following spontaneous transformation, both 2xSCS70 and 2xSCS2F10 cells grafted into nude mice gave rise to tumors containing Kit(+)cells and otherwise resembling gastrointestinal stromal tumors (GIST), which originate from the ICC lineage (Bardsley et al., Gastroenterology 2010;139:942-952). This study utilized oligonucleotide microarray analysis to charaterize the transcriptome of non-transformed 2xSCS70 and 2xSCS2F10 cells relative to their source tissues. Total RNA from ICC progenitor cell lines and their source tissue (gastric tunica muscularis from C57BL/6J mice (source of 2xSCS2F10 cells) and H-2Kb-tsA58 mice (source of 2xSCS70 cells) was isolated. Oligonucleotide micorarray studies were performed using 2-3 Affymetrix Mouse Genome 430.2 GeneChips per sample.

Project description:Interstitial cells of Cajal (ICC) are electrical pacemakers and mediators of neuromuscular neurotransmission in the gastrointestinal tract. Studies in organotypic cultures of intact gastric muscular wall tissues identified Kit(low)Cd44(+)Cd34(+)Insr(+)Igf1r(+) cells as local ICC progenitors (Lorincz et al., Gastroenterology 2008;134:1083-1093). Subsequently, conditionally immortalized Kit(low)Cd34(+) cells were isolated by immunomagnetic and fluorescent cell sorting and serial cloning from the gastric muscular wall of homozygous, 14-day-old H-2Kb-tsA58 mice and shown to share other phenotypic characteristics with in-situ ICC precursors (2xSCS70 cells; Bardsley et al., Gastroenterology 2010;139:942-952). 2xSCS70 cells were also found to express the ICC marker Ano1, to be able to extensively self-renew in the absence of the SV40 tsA58-TAg when cultured in NeuroCultÂ® Neural Stem Cell Proliferation Medium (NSCPM; STEMCELL Technologies) and to differentiate into Kit(+) ICC both in vitro and in vivo. Wild-type cells with Kit(low)Ano1(+)Cd44(+)Cd34(+)Insr(+)Igf1r(+) phenotype (2xSCS2F10 cells) were prospectively isolated from primary cultures of dissociated gastric muscular wall tissues from 7-day-old C57BL/6J mice by serial cloning following 150-day expansion in NSCPM containing 5% cell-free chick embryo extract (CEE; Accurate Chemical). Long-term culturing of both cell lines was associated with further decline in Kit expression while expression of Ano1 and other markers of ICC precursors was maintained. Following spontaneous transformation, both 2xSCS70 and 2xSCS2F10 cells grafted into nude mice gave rise to tumors containing Kit(+)cells and otherwise resembling gastrointestinal stromal tumors (GIST), which originate from the ICC lineage (Bardsley et al., Gastroenterology 2010;139:942-952). This study utilized RNA-sequencing to complement the companion microarray study in non-transformed 2xSCS70 and 2xSCS2F10 cells. Libraries were made from total RNA isolated from ICC progenitor cell lines using the Illumina TruSeq mRNA Sample Preparation Kit v2 and sequenced on the Illumina HiSeq 2000 platform.

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Cloning and Variation of Ground State Intestinal Stem Cells

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