{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/289436"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/289436","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Investigation of genetic PIK3CA activation in genome-edited human pluripotent stem cells","abstract":"Mosaic, activating mutations in PIK3CA, the gene encoding the catalytic p110α subunit of class IA phosphatidylinositol 3-kinase (PI3K), are the cause of rare, developmental growth disorders collectively known as PIK3CA-Related Overgrowth Spectrum (PROS). Given the pressing need for targeted therapy and evidence for tissue- and cell lineage-specific distribution of PIK3CA mutations in PROS, developmental models of this disease will be a key asset for preclinical drug testing and for a better understanding of PIK3CA activation in development. This PhD project addressed the lack of human, developmental PROS models by establishing isogenic series of human induced pluripotent stem cells (iPSCs) with endogenously expressed, activating PIK3CA mutations. This involved the optimisation of a CRISPR/Cas9 protocol for efficient knockin of different PIK3CA variants into human iPSCs. An isogenic iPSC series was established with cells expressing either wild-type PIK3CA or PIK3CA-H1047R, knocked into either one or both endogenous alleles. In parallel, mosaic patient- derived fibroblast cultures were reprogrammed to obtain isogenic wild-type and heterozygous iPSCs expressing PIK3CA-E418K. The models were used in comprehensive signalling studies, providing new insights into PI3K signalling in human iPSCs and how it is perturbed by genetic p110α activation. PIK3CA-E418K, a rare variant in both PROS and cancer, caused minimal pathway activation, in contrast to the highly recurrent variant PIK3CA-H1047R which induced strong PI3K signalling in both heterozygous and homozygous iPSCs according to a graded pattern. Studies of clinically relevant PI3K pathway inhibitors provided proof-of-concept that stem cell-based PROS models can be used for preclinical drug testing, and demonstrated that p110α is likely to be the main catalytic isoform mediating canonical PI3K signalling in human iPSCs. Differentiation assays revealed allele dose-dependent effects of PIK3CA-H1047R on stemness, with homozygous iPSCs exhibiting widespread transcriptome remodelling affect- ing genes implicated in cancer and development. Accordingly, these cells showed increased expression of pluripotency genes such as NANOG and NODAL, resulting in self-sustained “stemness” in embryoid body and teratoma assays. In comparison, heterozygous mutants behaved similar to wild-type controls under all differentiation paradigms. Furthermore, evidence was obtained that strong activation of PI3K signalling is fully compatible with definitive endoderm formation, arguing against cell-autonomous differentiation defects as the cause of endoderm sparing in PROS. In summary, these studies demonstrate the utility of human stem cell-based models of PROS for preclinical drug testing and for improved understanding of class IA PI3K signalling in human development. They are also likely to be useful in efforts to obtain a better understanding of PIK3CA-H1047R in human cancer.","abstract_html":"Mosaic, activating mutations in PIK3CA, the gene encoding the catalytic p110α subunit of class IA phosphatidylinositol 3-kinase (PI3K), are the cause of rare, developmental growth disorders collectively known as PIK3CA-Related Overgrowth Spectrum (PROS). Given the pressing need for targeted therapy and evidence for tissue- and cell lineage-specific distribution of PIK3CA mutations in PROS, developmental models of this disease will be a key asset for preclinical drug testing and for a better understanding of PIK3CA activation in development. This PhD project addressed the lack of human, developmental PROS models by establishing isogenic series of human induced pluripotent stem cells (iPSCs) with endogenously expressed, activating PIK3CA mutations. This involved the optimisation of a CRISPR/Cas9 protocol for efficient knockin of different PIK3CA variants into human iPSCs. An isogenic iPSC series was established with cells expressing either wild-type PIK3CA or PIK3CA-H1047R, knocked into either one or both endogenous alleles. In parallel, mosaic patient- derived fibroblast cultures were reprogrammed to obtain isogenic wild-type and heterozygous iPSCs expressing PIK3CA-E418K. The models were used in comprehensive signalling studies, providing new insights into PI3K signalling in human iPSCs and how it is perturbed by genetic p110α activation. PIK3CA-E418K, a rare variant in both PROS and cancer, caused minimal pathway activation, in contrast to the highly recurrent variant PIK3CA-H1047R which induced strong PI3K signalling in both heterozygous and homozygous iPSCs according to a graded pattern. Studies of clinically relevant PI3K pathway inhibitors provided proof-of-concept that stem cell-based PROS models can be used for preclinical drug testing, and demonstrated that p110α is likely to be the main catalytic isoform mediating canonical PI3K signalling in human iPSCs. Differentiation assays revealed allele dose-dependent effects of PIK3CA-H1047R on stemness, with homozygous iPSCs exhibiting widespread transcriptome remodelling affect- ing genes implicated in cancer and development. Accordingly, these cells showed increased expression of pluripotency genes such as NANOG and NODAL, resulting in self-sustained “stemness” in embryoid body and teratoma assays. In comparison, heterozygous mutants behaved similar to wild-type controls under all differentiation paradigms. Furthermore, evidence was obtained that strong activation of PI3K signalling is fully compatible with definitive endoderm formation, arguing against cell-autonomous differentiation defects as the cause of endoderm sparing in PROS. In summary, these studies demonstrate the utility of human stem cell-based models of PROS for preclinical drug testing and for improved understanding of class IA PI3K signalling in human development. They are also likely to be useful in efforts to obtain a better understanding of PIK3CA-H1047R in human cancer.","abstract_has_math":false,"creators":["Madsen, Ralitsa Radostinova"],"institution":"University of Cambridge","degree_name":"PhD","degree_level":"doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Semple, Robert Kenneth"],"committee_chairs":[],"committee_members":[],"year":2019,"date_issued":"2019-05-18","date_published":"2019-05-18","updated_at":"2026-07-22T22:24:28Z","subjects":["signal transduction","PI3K","PROS","human pluripotent stem cells","development","cancer","phosphatidylinositol 3-kinase","PIK3CA","CRISPR"],"languages":["en"],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/46128253-de5a-446e-a9c5-7a4ea51f4c2f/download","https://creativecommons.org/licenses/by-nc-nd/4.0/"],"identifier_entries":[{"key":"dc:creator.authoridentifier","label":"Author Identifier","values":["0000000188445167","0000000165393069"],"render_values":[{"text":"0000-0001-8844-5167","href":"https://orcid.org/0000-0001-8844-5167","code":true},{"text":"0000-0001-6539-3069","href":"https://orcid.org/0000-0001-6539-3069","code":true}]}]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.35662","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Semple, Robert Kenneth"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["Wellcome Trust 4-year PhD Studentship"]},{"key":"dc:creator","label":"Author","values":["Madsen, Ralitsa Radostinova"]},{"key":"dc:creator.authoridentifier","label":"Author Identifier","values":["0000000188445167","0000000165393069"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2019-05-18"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/289436"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["PhD"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["signal transduction","PI3K","PROS","human pluripotent stem cells","development","cancer","phosphatidylinositol 3-kinase","PIK3CA","CRISPR"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/46128253-de5a-446e-a9c5-7a4ea51f4c2f/download","https://creativecommons.org/licenses/by-nc-nd/4.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["10.17863/CAM.35662"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/fe1820f3-8cfb-4fa5-b7f8-60db8a679653/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Mosaic, activating mutations in PIK3CA, the gene encoding the catalytic p110α subunit of class IA phosphatidylinositol 3-kinase (PI3K), are the cause of rare, developmental growth disorders collectively known as PIK3CA-Related Overgrowth Spectrum (PROS). Given the pressing need for targeted therapy and evidence for tissue- and cell lineage-specific distribution of PIK3CA mutations in PROS, developmental models of this disease will be a key asset for preclinical drug testing and for a better understanding of PIK3CA activation in development. This PhD project addressed the lack of human, developmental PROS models by establishing isogenic series of human induced pluripotent stem cells (iPSCs) with endogenously expressed, activating PIK3CA mutations. This involved the optimisation of a CRISPR/Cas9 protocol for efficient knockin of different PIK3CA variants into human iPSCs. An isogenic iPSC series was established with cells expressing either wild-type PIK3CA or PIK3CA-H1047R, knocked into either one or both endogenous alleles. In parallel, mosaic patient- derived fibroblast cultures were reprogrammed to obtain isogenic wild-type and heterozygous iPSCs expressing PIK3CA-E418K. The models were used in comprehensive signalling studies, providing new insights into PI3K signalling in human iPSCs and how it is perturbed by genetic p110α activation. PIK3CA-E418K, a rare variant in both PROS and cancer, caused minimal pathway activation, in contrast to the highly recurrent variant PIK3CA-H1047R which induced strong PI3K signalling in both heterozygous and homozygous iPSCs according to a graded pattern. Studies of clinically relevant PI3K pathway inhibitors provided proof-of-concept that stem cell-based PROS models can be used for preclinical drug testing, and demonstrated that p110α is likely to be the main catalytic isoform mediating canonical PI3K signalling in human iPSCs. Differentiation assays revealed allele dose-dependent effects of PIK3CA-H1047R on stemness, with homozygous iPSCs exhibiting widespread transcriptome remodelling affect- ing genes implicated in cancer and development. Accordingly, these cells showed increased expression of pluripotency genes such as NANOG and NODAL, resulting in self-sustained “stemness” in embryoid body and teratoma assays. In comparison, heterozygous mutants behaved similar to wild-type controls under all differentiation paradigms. Furthermore, evidence was obtained that strong activation of PI3K signalling is fully compatible with definitive endoderm formation, arguing against cell-autonomous differentiation defects as the cause of endoderm sparing in PROS. In summary, these studies demonstrate the utility of human stem cell-based models of PROS for preclinical drug testing and for improved understanding of class IA PI3K signalling in human development. They are also likely to be useful in efforts to obtain a better understanding of PIK3CA-H1047R in human cancer."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["5d4b406e4f200467c91a9d90d777c753","87eda9de84448d1f82354d60eee3eb5f"]},{"key":"dc:title","label":"Title","values":["Investigation of genetic PIK3CA activation in genome-edited human pluripotent stem cells"]}]}],"canonical_facts":{"dc:contributor.advisor":["Semple, Robert Kenneth"],"dc:contributor.sponsor":["Wellcome Trust 4-year PhD Studentship"],"dc:creator":["Madsen, Ralitsa Radostinova"],"dc:creator.authoridentifier":["0000000188445167","0000000165393069"],"dc:date.issued":["2019-05-18"],"dc:description.abstract":["Mosaic, activating mutations in PIK3CA, the gene encoding the catalytic p110α subunit of class IA phosphatidylinositol 3-kinase (PI3K), are the cause of rare, developmental growth disorders collectively known as PIK3CA-Related Overgrowth Spectrum (PROS). Given the pressing need for targeted therapy and evidence for tissue- and cell lineage-specific distribution of PIK3CA mutations in PROS, developmental models of this disease will be a key asset for preclinical drug testing and for a better understanding of PIK3CA activation in development. This PhD project addressed the lack of human, developmental PROS models by establishing isogenic series of human induced pluripotent stem cells (iPSCs) with endogenously expressed, activating PIK3CA mutations. This involved the optimisation of a CRISPR/Cas9 protocol for efficient knockin of different PIK3CA variants into human iPSCs. An isogenic iPSC series was established with cells expressing either wild-type PIK3CA or PIK3CA-H1047R, knocked into either one or both endogenous alleles. In parallel, mosaic patient- derived fibroblast cultures were reprogrammed to obtain isogenic wild-type and heterozygous iPSCs expressing PIK3CA-E418K. The models were used in comprehensive signalling studies, providing new insights into PI3K signalling in human iPSCs and how it is perturbed by genetic p110α activation. PIK3CA-E418K, a rare variant in both PROS and cancer, caused minimal pathway activation, in contrast to the highly recurrent variant PIK3CA-H1047R which induced strong PI3K signalling in both heterozygous and homozygous iPSCs according to a graded pattern. Studies of clinically relevant PI3K pathway inhibitors provided proof-of-concept that stem cell-based PROS models can be used for preclinical drug testing, and demonstrated that p110α is likely to be the main catalytic isoform mediating canonical PI3K signalling in human iPSCs. Differentiation assays revealed allele dose-dependent effects of PIK3CA-H1047R on stemness, with homozygous iPSCs exhibiting widespread transcriptome remodelling affect- ing genes implicated in cancer and development. Accordingly, these cells showed increased expression of pluripotency genes such as NANOG and NODAL, resulting in self-sustained “stemness” in embryoid body and teratoma assays. In comparison, heterozygous mutants behaved similar to wild-type controls under all differentiation paradigms. Furthermore, evidence was obtained that strong activation of PI3K signalling is fully compatible with definitive endoderm formation, arguing against cell-autonomous differentiation defects as the cause of endoderm sparing in PROS. In summary, these studies demonstrate the utility of human stem cell-based models of PROS for preclinical drug testing and for improved understanding of class IA PI3K signalling in human development. They are also likely to be useful in efforts to obtain a better understanding of PIK3CA-H1047R in human cancer."],"dc:format.checksum.md5":["5d4b406e4f200467c91a9d90d777c753","87eda9de84448d1f82354d60eee3eb5f"],"dc:identifier.doi":["10.17863/CAM.35662"],"dc:identifier.uri":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/fe1820f3-8cfb-4fa5-b7f8-60db8a679653/download"],"dc:language":["en"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/289436"],"dc:rights":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/46128253-de5a-446e-a9c5-7a4ea51f4c2f/download","https://creativecommons.org/licenses/by-nc-nd/4.0/"],"dc:subject":["signal transduction","PI3K","PROS","human pluripotent stem cells","development","cancer","phosphatidylinositol 3-kinase","PIK3CA","CRISPR"],"dc:title":["Investigation of genetic PIK3CA activation in genome-edited human pluripotent stem cells"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["doctoral"],"dc:type.qualificationname":["PhD"]},"updated_at":"2026-07-22T22:24:28Z"}