Human pluripotent stem cells and CRISPR-Cas9 genome editing to model diabetes

Research output: ThesisDoctoral ThesisCollection of Articles

Abstract

Pancreatic beta-cell dysfunction is the ultimate cause behind all forms of diabetes. Decades of research with different animal and cellular models have expanded the knowledge on the heterogeneous molecular mechanisms causing the disease. However, they present important limitations that may significantly affect the way these findings can be translated into new approaches to combat diabetes in humans. Rodent pancreatic islet development and physiology display species-specific particularities when compared to human. Similarly, rodent and human insulinoma cell lines are a convenient research tool but do not recapitulate faithfully the functionality of adult human beta-cells. To validate if the findings obtained with these models extrapolate to humans, diabetes researchers have traditionally used cadaveric donor human islets. Primary islets are scarce, highly variable in their composition and functionality and difficult to manipulate for certain experiments. As an alternative, human pluripotent stem cells (hPSC) constitute a renewable source of beta-cells. Stem cell-derived beta-cells can be generated by directed differentiation and used as a model to study pancreatic beta-cell development and disease in vitro. They can also be transplanted into immunocompromised mice, generating humanized models where in vivo beta-cell function can be closely evaluated in a systemic context. The goal of this thesis work was to demonstrate the use of human pluripotent stem cells as a tool to investigate monogenic diabetes disease mechanisms. For this purpose, improved hPSC differentiation protocols to the beta-cell lineage were generated utilizing 3D suspension culture approaches. Transplantation procedures were devised to create humanized mouse models that allow proper evaluation of beta-cell function in vivo. Novel CRISPR-Cas9-based techniques were established and utilized to edit the genome of hPSC and control gene transcription. Precise genome editing made possible the generation of isogenic, mutation-corrected patient-derived induced PSC, enabling the disease modeling of monogenic diabetes cases. Using these approaches, an activating mutation in STAT3 gene was found to cause neonatal diabetes by inducing pancreas endocrinogenesis prematurely, via direct induction of master endocrine transcription factor NEUROG3. In a similar way, INS gene mutations causing proinsulin misfolding were found to impair developing beta-cell proliferation due to increased endoplasmic reticulum stress. Taken together, this thesis work highlights the versatility of hPSC combined with genome editing and transplantation as a useful approach to better elucidate and understand human diabetes.
Original languageEnglish
Supervisors/Advisors
  • Otonkoski, Timo, Supervisor
Place of PublicationHelsinki
Publisher
Print ISBNs978-951-51-4437-9
Electronic ISBNs 978-951-51-4438-6
Publication statusPublished - 2018
MoE publication typeG5 Doctoral dissertation (article)

Fields of Science

  • Pluripotent Stem Cells
  • Diabetes Mellitus
  • +etiology
  • Cells, Cultured
  • Insulin-Secreting Cells
  • Islets of Langerhans
  • Mice
  • CRISPR-Cas Systems
  • STAT3 Transcription Factor
  • +genetics
  • Proinsulin
  • Gene Editing
  • 3111 Biomedicine

Cite this

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title = "Human pluripotent stem cells and CRISPR-Cas9 genome editing to model diabetes",
abstract = "Pancreatic beta-cell dysfunction is the ultimate cause behind all forms of diabetes. Decades of research with different animal and cellular models have expanded the knowledge on the heterogeneous molecular mechanisms causing the disease. However, they present important limitations that may significantly affect the way these findings can be translated into new approaches to combat diabetes in humans. Rodent pancreatic islet development and physiology display species-specific particularities when compared to human. Similarly, rodent and human insulinoma cell lines are a convenient research tool but do not recapitulate faithfully the functionality of adult human beta-cells. To validate if the findings obtained with these models extrapolate to humans, diabetes researchers have traditionally used cadaveric donor human islets. Primary islets are scarce, highly variable in their composition and functionality and difficult to manipulate for certain experiments. As an alternative, human pluripotent stem cells (hPSC) constitute a renewable source of beta-cells. Stem cell-derived beta-cells can be generated by directed differentiation and used as a model to study pancreatic beta-cell development and disease in vitro. They can also be transplanted into immunocompromised mice, generating humanized models where in vivo beta-cell function can be closely evaluated in a systemic context. The goal of this thesis work was to demonstrate the use of human pluripotent stem cells as a tool to investigate monogenic diabetes disease mechanisms. For this purpose, improved hPSC differentiation protocols to the beta-cell lineage were generated utilizing 3D suspension culture approaches. Transplantation procedures were devised to create humanized mouse models that allow proper evaluation of beta-cell function in vivo. Novel CRISPR-Cas9-based techniques were established and utilized to edit the genome of hPSC and control gene transcription. Precise genome editing made possible the generation of isogenic, mutation-corrected patient-derived induced PSC, enabling the disease modeling of monogenic diabetes cases. Using these approaches, an activating mutation in STAT3 gene was found to cause neonatal diabetes by inducing pancreas endocrinogenesis prematurely, via direct induction of master endocrine transcription factor NEUROG3. In a similar way, INS gene mutations causing proinsulin misfolding were found to impair developing beta-cell proliferation due to increased endoplasmic reticulum stress. Taken together, this thesis work highlights the versatility of hPSC combined with genome editing and transplantation as a useful approach to better elucidate and understand human diabetes.",
keywords = "Pluripotent Stem Cells, Diabetes Mellitus, +etiology, Cells, Cultured, Insulin-Secreting Cells, Islets of Langerhans, Mice, CRISPR-Cas Systems, STAT3 Transcription Factor, +genetics, Proinsulin, Gene Editing, 3111 Biomedicine",
author = "{Balboa Alonso}, Diego",
note = "M1 - 164 s. + liitteet",
year = "2018",
language = "English",
isbn = "978-951-51-4437-9",
series = "Dissertationes Scholae Doctoralis Ad Sanitatem Investigandam Universitatis Helsinkiensis",
publisher = "Helsingin yliopisto",
number = "54/2018",
address = "Finland",

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Human pluripotent stem cells and CRISPR-Cas9 genome editing to model diabetes. / Balboa Alonso, Diego.

Helsinki : Helsingin yliopisto, 2018. 164 p.

Research output: ThesisDoctoral ThesisCollection of Articles

TY - THES

T1 - Human pluripotent stem cells and CRISPR-Cas9 genome editing to model diabetes

AU - Balboa Alonso, Diego

N1 - M1 - 164 s. + liitteet

PY - 2018

Y1 - 2018

N2 - Pancreatic beta-cell dysfunction is the ultimate cause behind all forms of diabetes. Decades of research with different animal and cellular models have expanded the knowledge on the heterogeneous molecular mechanisms causing the disease. However, they present important limitations that may significantly affect the way these findings can be translated into new approaches to combat diabetes in humans. Rodent pancreatic islet development and physiology display species-specific particularities when compared to human. Similarly, rodent and human insulinoma cell lines are a convenient research tool but do not recapitulate faithfully the functionality of adult human beta-cells. To validate if the findings obtained with these models extrapolate to humans, diabetes researchers have traditionally used cadaveric donor human islets. Primary islets are scarce, highly variable in their composition and functionality and difficult to manipulate for certain experiments. As an alternative, human pluripotent stem cells (hPSC) constitute a renewable source of beta-cells. Stem cell-derived beta-cells can be generated by directed differentiation and used as a model to study pancreatic beta-cell development and disease in vitro. They can also be transplanted into immunocompromised mice, generating humanized models where in vivo beta-cell function can be closely evaluated in a systemic context. The goal of this thesis work was to demonstrate the use of human pluripotent stem cells as a tool to investigate monogenic diabetes disease mechanisms. For this purpose, improved hPSC differentiation protocols to the beta-cell lineage were generated utilizing 3D suspension culture approaches. Transplantation procedures were devised to create humanized mouse models that allow proper evaluation of beta-cell function in vivo. Novel CRISPR-Cas9-based techniques were established and utilized to edit the genome of hPSC and control gene transcription. Precise genome editing made possible the generation of isogenic, mutation-corrected patient-derived induced PSC, enabling the disease modeling of monogenic diabetes cases. Using these approaches, an activating mutation in STAT3 gene was found to cause neonatal diabetes by inducing pancreas endocrinogenesis prematurely, via direct induction of master endocrine transcription factor NEUROG3. In a similar way, INS gene mutations causing proinsulin misfolding were found to impair developing beta-cell proliferation due to increased endoplasmic reticulum stress. Taken together, this thesis work highlights the versatility of hPSC combined with genome editing and transplantation as a useful approach to better elucidate and understand human diabetes.

AB - Pancreatic beta-cell dysfunction is the ultimate cause behind all forms of diabetes. Decades of research with different animal and cellular models have expanded the knowledge on the heterogeneous molecular mechanisms causing the disease. However, they present important limitations that may significantly affect the way these findings can be translated into new approaches to combat diabetes in humans. Rodent pancreatic islet development and physiology display species-specific particularities when compared to human. Similarly, rodent and human insulinoma cell lines are a convenient research tool but do not recapitulate faithfully the functionality of adult human beta-cells. To validate if the findings obtained with these models extrapolate to humans, diabetes researchers have traditionally used cadaveric donor human islets. Primary islets are scarce, highly variable in their composition and functionality and difficult to manipulate for certain experiments. As an alternative, human pluripotent stem cells (hPSC) constitute a renewable source of beta-cells. Stem cell-derived beta-cells can be generated by directed differentiation and used as a model to study pancreatic beta-cell development and disease in vitro. They can also be transplanted into immunocompromised mice, generating humanized models where in vivo beta-cell function can be closely evaluated in a systemic context. The goal of this thesis work was to demonstrate the use of human pluripotent stem cells as a tool to investigate monogenic diabetes disease mechanisms. For this purpose, improved hPSC differentiation protocols to the beta-cell lineage were generated utilizing 3D suspension culture approaches. Transplantation procedures were devised to create humanized mouse models that allow proper evaluation of beta-cell function in vivo. Novel CRISPR-Cas9-based techniques were established and utilized to edit the genome of hPSC and control gene transcription. Precise genome editing made possible the generation of isogenic, mutation-corrected patient-derived induced PSC, enabling the disease modeling of monogenic diabetes cases. Using these approaches, an activating mutation in STAT3 gene was found to cause neonatal diabetes by inducing pancreas endocrinogenesis prematurely, via direct induction of master endocrine transcription factor NEUROG3. In a similar way, INS gene mutations causing proinsulin misfolding were found to impair developing beta-cell proliferation due to increased endoplasmic reticulum stress. Taken together, this thesis work highlights the versatility of hPSC combined with genome editing and transplantation as a useful approach to better elucidate and understand human diabetes.

KW - Pluripotent Stem Cells

KW - Diabetes Mellitus

KW - +etiology

KW - Cells, Cultured

KW - Insulin-Secreting Cells

KW - Islets of Langerhans

KW - Mice

KW - CRISPR-Cas Systems

KW - STAT3 Transcription Factor

KW - +genetics

KW - Proinsulin

KW - Gene Editing

KW - 3111 Biomedicine

M3 - Doctoral Thesis

SN - 978-951-51-4437-9

T3 - Dissertationes Scholae Doctoralis Ad Sanitatem Investigandam Universitatis Helsinkiensis

PB - Helsingin yliopisto

CY - Helsinki

ER -

Balboa Alonso D. Human pluripotent stem cells and CRISPR-Cas9 genome editing to model diabetes. Helsinki: Helsingin yliopisto, 2018. 164 p. (Dissertationes Scholae Doctoralis Ad Sanitatem Investigandam Universitatis Helsinkiensis ; 54/2018).