Directed Differentiation of Basal Forebrain Cholinergic Neurons From Human Pluripotent Stem Cells

J Neurosci Methods. 2016 Jun 15;266:42-9. doi: 10.1016/j.jneumeth.2016.03.017. Epub 2016 Mar 29.

Abstract

Background: Basal forebrain cholinergic neurons (BFCNs) play critical roles in learning, memory and cognition. Dysfunction or degeneration of BFCNs may connect to neuropathology, such as Alzheimer's disease, Down's syndrome and dementia. Generation of functional BFCNs may contribute to the studies of cell-based therapy and pathogenesis that is related to learning and memory deficits.

New method: Here we describe a detail method for robust generation of BFCNs from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). In this method, BFCN progenitors are patterned from hESC or hiPSC-derived primitive neuroepithelial cells, with the treatment of sonic hedgehog (SHH) or combination with its agonist Purmorphamine, and by co-culturing with human astrocytes.

Results: At day 20, ∼90% hPSC-derived progenitors expressed NKX2.1, which is a transcriptional marker for MGE. Moreover, around 40% of NKX2.1+ cells co-expressed OLIG2 and ∼15% of NKX2.1+ cells co-expressed ISLET1, which are ventral markers. At day 35, ∼40% neurons robustly express ChAT, most of which are co-labeled with NKX2.1, ISLET1 and FOXG1, indicating the basal forebrain-like identity. At day 45, these neurons express mature neuronal markers MAP2, Synapsin, and VAChT.

Comparison with existing method(s): In this method, undefined conditions including genetic modification or cell-sorting are avoided. As a choice, feeder free conditions are used to avoid ingredients of animal origin. Moreover, Purmorphamine can be substituted for SHH to induce ventral progenitors effectively and economically.

Conclusion: We provide an efficient method to generate BFCNs from multiple hPSC lines, which offers the potential application for disease modeling and pharmacological studies.

Keywords: Basal forebrain cholinergic neurons; Human embryonic stem cells; Human induced pluripotent stem cells; Medial ganglionic eminence; Neural differentiation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / metabolism
  • Astrocytes / cytology
  • Astrocytes / physiology
  • Basal Forebrain / cytology
  • Basal Forebrain / physiology*
  • Cell Culture Techniques / instrumentation
  • Cell Culture Techniques / methods*
  • Cell Line
  • Choline O-Acetyltransferase / metabolism
  • Coculture Techniques / instrumentation
  • Coculture Techniques / methods
  • Embryonic Stem Cells / cytology
  • Embryonic Stem Cells / physiology*
  • Forkhead Transcription Factors / metabolism
  • Humans
  • Induced Pluripotent Stem Cells / cytology
  • Induced Pluripotent Stem Cells / physiology*
  • Nerve Tissue Proteins / metabolism
  • Nestin / metabolism
  • Neurogenesis / physiology*
  • Neurons / cytology
  • Neurons / physiology*
  • PAX6 Transcription Factor / metabolism
  • SOXB1 Transcription Factors / metabolism
  • Thyroid Nuclear Factor 1 / metabolism

Substances

  • Adaptor Proteins, Signal Transducing
  • FOXG1 protein, human
  • Forkhead Transcription Factors
  • MAPK8IP1 protein, human
  • NES protein, human
  • NKX2-1 protein, human
  • Nerve Tissue Proteins
  • Nestin
  • PAX6 Transcription Factor
  • PAX6 protein, human
  • SOX1 protein, human
  • SOX2 protein, human
  • SOXB1 Transcription Factors
  • Thyroid Nuclear Factor 1
  • Choline O-Acetyltransferase