Home › Cell Biology › Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts
Cell Biology JoVE (Open Access) Citable · DOI

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

DOI: 10.3791/56904-v
What you'll learn
  • ✓ Prepare and plate adult human dermal fibroblasts for direct neuronal reprogramming
  • ✓ Perform viral transduction and monitor cell conversion over multiple days
  • ✓ Isolate induced neurons using FACS based on neuronal markers
  • ✓ Generate functional neurons from adult human skin cells in high yield
Protocol

Biopharma Insights Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Difficulty
advanced
Total time
~7-10 days (including viral transduction, conversion period, and FACS isolation)
Model organism
Human adult dermal fibroblasts
Biosafety
BSL-1

Steps

1
Prepare skin fibroblasts for reprogramming

Isolate and culture adult human dermal fibroblasts from donor skin samples. Prepare cells in optimal condition for subsequent reprogramming protocol.

▶ 01:02
2
Plate fibroblasts for reprogramming on day one

Seed prepared fibroblasts at appropriate density on culture dishes to establish baseline population before viral transduction.

▶ 01:43
3
Perform viral transduction of fibroblasts

Apply single vector-based viral construct to fibroblasts to initiate direct neuronal reprogramming on day zero.

▶ 03:22
4
Maintain converting cells over culture period

Monitor and support converting fibroblasts through media changes and culture maintenance during multi-day reprogramming process.

▶ 04:12
5
Isolate induced neurons using fluorescence-activated cell sorting

Perform FACS to separate successfully reprogrammed induced neurons from unconverted fibroblasts using neuronal marker expression.

▶ 04:58
6
Analyze results and verify neuronal identity

Confirm successful generation of induced neurons from adult human dermal fibroblasts and assess yield and quality of reprogrammed cells.

▶ 07:33
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