Home › Genetics / Genomics › Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development
Genetics / Genomics JoVE (Open Access) Citable · DOI

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

DOI: 10.3791/55267-v
What you'll learn
  • ✓ Generate hPSC mutant lines using the iCRISPR genome editing platform
  • ✓ Differentiate hPSCs into glucose-responsive pancreatic β-like cells
  • ✓ Apply combined genome editing and directed differentiation to study lineage determinants
  • ✓ Analyze genetic modifications for human pancreatic development and disease models
Protocol

Protocols to generate hPSC mutant lines using the iCRISPR platform and to differentiate hPSCs into glucose-responsive β-like cells are described. Combining genome editing technology with hPSC-directed differentiation provides a powerful platform for the systematic analysis of the role of lineage determinants in human development and disease progression.

Difficulty
advanced
Total time
~4–6 weeks (including hPSC generation, gRNA transfection, selection, and differentiation protocol)
Model organism
Human pluripotent stem cells (hPSCs)
Biosafety
BSL-1

Steps

1
Generate iCas9 hPSC lines

Establish stable hPSC lines expressing inducible Cas9 (iCas9) for controlled CRISPR/Cas9 genome editing. This foundational step creates the genetic background required for subsequent targeted modifications.

▶ 00:52
2
Transfect guide RNA into iCas9 hPSCs

Introduce specific guide RNAs (gRNAs) into established iCas9 hPSC lines to direct Cas9-mediated genome editing at target loci. This step enables precise genetic modifications of lineage determinant genes.

▶ 04:15
3
Differentiate hPSCs into glucose-responsive β cells

Apply directed differentiation protocols to drive hPSCs (wild-type and edited lines) through pancreatic lineage commitment into functional, glucose-responsive insulin-secreting β-like cells. This step generates the target cell type for functional analysis.

▶ 06:00
4
Analyze genetic modification efficiency and outcomes

Assess successful genome editing in hPSCs and characterize resulting β-like cells for gene expression, glucose responsiveness, and phenotypic changes. Results demonstrate platform efficacy for interrogating lineage determinants.

▶ 07:48
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