Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Summary

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

Abstract

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-β and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Introduction

As opposed to reprogramming of somatic cells into induced pluripotent stem cells (iPSCs), which in turn are endowed with a plethora of differentiation potentials, direct reprogramming aims for straight conversion of one specific cell type into another. With respect to their application in the context of disease modeling and potential cell-based therapies, both reprogramming approaches have specific advantages and disadvantages. Reprogramming into iPSCs (i) provides a virtually infinite source of cells; (ii) allows for genetic engineering; (iii) endows with a nearly unlimited differentiation potential. However, major disadvantages of iPSCs entail the risk of tumorigeni....

Protocol

1. Isolation and Culturing of Adult Human Brain Cells

Experiments involving human tissue should be performed in accordance with all relevant governmental and institutional regulations regarding the use of human material for research purposes. The present protocol was developed in accordance with the approval by the ethical committee of the Medical Faculty of the LMU Munich and written informed consent from all patients.

This protocol of preparing cultures of the human adult cerebral cortex has been established using specimen of patients of both sexes suffering from temporal lobe epilepsy or other deep-seated non-tr....

Results

The first outcome of this protocol after successfully establishing a culture from a specimen of human adult cerebral cortex consists in the identification of the cellular composition of the culture. Immunocytochemistry for cell type specific proteins reveals a considerable degree of heterogeneity between cultures derived from specimen of different patients (Figure 1A). As quantified by flow cytometry there is always a substantial fraction of cells that express PDGFRβ (on average ~75%, ranging from 30 to .......

Discussion

The present protocol describes the in vitro expansion and enrichment of pericyte-derived cells following isolation from the adult human cerebral cortex and the subsequent conversion into iNs by retrovirus-mediated expression of the neurogenic transcription factors Sox2 and Ascl1. Such protocol provides an experimental in vitro system to study the lineage-conversion of brain-resident cells into neurons and potentially also glia, with the goal in mind to ultimately translate this direct conversion approac.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
anti-CD140b-PE	BD Biosciences	558821	use 1:100
anti-CD146-FITC	AbD Serotec	MCA2141FT	use 1:100
anti-CD13-FITC	AbD Serotec	MVA1270A488T	use 1:100
anti-CD34-APC	BD Biosciences	560940	use 1:100
anti-PDGFRβ	Cell Signaling	3169S	use 1:200
anti-CD146	Abcam	Ab75769	use 1:400
anti-NG2	Millipore	AB5320	use 1:400
anti-SMA	Sigma-Aldrich	A2547	use 1:400
anti-βIII-tubulin	Sigma-Aldrich	T8660	use 1:400
anti-MAP2	Sigma-Aldrich	M4403	use 1:200
anti-GFAP	Sigma-Aldrich	G3893	use 1:600
anti-GFP	Aves Labs	GFP 10-20	use 1:1,000
anti-RFP	Chromotek	5F8	use 1:500
anti-S100β	Sigma-Aldrich	S2532	use 1:300
anti-NeuN	Millipore	MAB377	use 1:200
anti-GABA	Sigma-Aldrich	A2052	use 1:500
anti-Calretinin	Millipore	AB5054	use 1:500
anti-vGluT1	SYnaptic SYstems	135511	use 1:1,000
Rat IgG1 isotype control FITC	AbD Serotec	11-4301-81	use 1:100
Rat IgG1 isotype control PE	AbD Serotec	12-4301-81	use 1:100
Rat IgG1 isotype control APC	AbD Serotec	17-4301-81	use 1:100
TrypLE	Invitrogen	12605-010
DMEM, high glucose, GlutaMAX	Invitrogen	61965
B27 serum-free supplement	Invitrogen	17504044
fetal calf serum	Invitrogen	10106-169	perform heat inactivation by incubation at 56 °C for 30 min
Penicillin-streptomycin	Invitrogen	15140122
HBSS (Hanks Balanced Salt Solution)	Invitrogen	24020-091
Dulbecco's PBS without CaCl2 and MgCl2 (D-PBS, 1x)	Invitrogen	14190
HEPES	Sigma-Aldrich	H3375
Poly-D-lysine hydrobromide (PDL)	Sigma-Aldrich	P0899
PFA (paraformaldehyde)	Sigma-Aldrich	P1648
Bovine serum albumin	Sigma-Aldrich	A9418
Triton X-100	Sigma-Aldrich	T9284
DAPI (4′,6-Diamidin-2-phenylindole) dilactate	Sigma-Aldrich	042M4005
Aqua-Poly/Mount	Polysciences	18606-20
Polypropylene round-bottom tubes	BD Biosciences	352063
Polystyrene round-bottom tubes with cell-strainer cap	BD Biosciences	352235
24-well plates	Orange Scientific	5530305
75 cm2 culture flasks	Greiner Bio-One	658175
25 cm2 culture flasks	Greiner Bio-One	690175
Disposable pipettes 10 ml	Sarstedt	86.1254.001
Glass Pasteur pipettes (150 mm)	Fisherbrand	FB50251
Glass coverslips 12 mm diameter	Menzel	CB00120RA1
Surgical disposable scalpels	B. Braun	5518083
Tissue culture dishes	Greiner Bio-One	633180
Conical tubes 15 ml	BD Biosciences	352095
Laminar flow hood
Centrifuge and swing-out rotor with adapters for 15 ml and 50 ml tubes	Hettich Lab Technology	1406
Flow cytometry cell sorter: FACSAria l with FACSDiva software	BD Biosciences
Humified cell culture incubator	Eppendorf Galaxy 170R
Wather bath at 37 °C
FACSFlow sheath fluid	BD Biosciences	342003
Epifluorescence microscope BX61	Olympus
Confocal microscope LSM710	Zeiss