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 tumorigenicity of the undifferentiated cells (teratoma formation in vivo) and the need for ex vivo cultivation and subsequent transplantation if these cells are to be used for cell-based therapies. Conversely, direct lineage-reprogramming is restricted by the lower yield of the desired cells which correlates directly with the number of the targeted cells of the starting population, but possesses the advantage that lineage-reprogrammed cells appear to exhibit no tumorigenic risk upon transplantation^1,2; furthermore, direct reprogramming can even be achieved in situ, i.e. within the organ where these cells would be required, thus avoiding the need of transplantation.

With this in mind, our lab has pursued the possibility of lineage-reprogramming brain-resident cells into iNs as a novel approach towards cell-based therapies of neurodegenerative diseases. Brain-resident cells that may be potentially considered as cellular targets for lineage-reprogramming comprise different types of macroglia (astrocytes, NG2 cells and oligodendrocytes), microglia, and microvessel-associated cells (endothelial cells and pericytes). We have extensively studied the in vitro reprogramming potential of astroglia of the cerebral cortex of early postnatal mice^3-5. In search of similarly suitable cell sources for direct lineage-reprogramming in the adult human brain, we encountered a cell population that can be successfully reprogrammed into iNs and exhibit hallmarks of pericytes. Here we describe a protocol of how to harvest these cells from adult human brain biopsies, to expand and enrich these cells in vitro, and finally successfully reprogram a substantial fraction of these in vitro expanded cells (in the range of 25-30%) into iNs. Reprogramming can be achieved by simultaneous retrovirus-mediated co-expression of two transcription factors, sox2 and ascl1. These PdiNs were found to acquire the ability of repetitive action potential firing and to serve as synaptic targets for other neurons indicating their capability of integrating into neural networks. Our protocol provides a straightforward procedure for the isolation and lineage conversion of adult human brain pericytes into iNs.

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-traumatic, non-malignant lesions. The tissue obtained from the surgical room comprised exclusively the access channel to the brain lesion and therefore is considered healthy. The age range of the patients was 19-70 years.

1. Prepare growth medium by adding heat-inactivated fetal calf serum (FCS) to DMEM high glucose with GlutaMAX to obtain a final concentration of 20% FCS. Add 5 ml penicillin/streptomycin to a total of 500 ml growth medium. Perform this and all subsequent steps requiring sterile culture conditions in an appropriate laminar flow hood.
2. Keep the adult human brain biopsy obtained from the surgical room in Hanks’ balanced salt solution with CaCl₂ and MgCl₂ (HBSS) medium including HEPES (10 mM final concentration) on ice until processing. Start processing as soon as possible.
3. To start the dissociation into single cells, transfer the tissue into a 65 mm petri dish and mince into small pieces by using two sterile single-use scalpels. For enzymatic digestion use 3-6 ml TrypLE in a 15 ml conical tube and incubate for 15-30 min at 37 °C in a water bath.
4. Add 1 volume of prewarmed growth medium to facilitate dissociation and gently triturate the solution containing tissue pieces up and down by first using a 5 ml disposable pipette, followed by using a glass Pasteur pipette until homogenization of the cell suspension. Typically, some residual tissue pieces, mostly consisting of white matter, will remain in the suspension.
5. Spin down at 157 x g for 5 min and resuspend the pellet in the appropriate amount of growth medium (10 ml per uncoated T75 culture flask). Use one T75 culture flask for a biopsy of 5-10 mm diameter in size and extrapolate from this in case of larger specimens.
6. Place cells into a 37 °C incubator with 5% CO₂ and 5% O₂.
7. Replace half of the medium every 3-4 days until cells have reached confluency. This usually takes up to two weeks (referred to as passage 0 [P0]).

2. In vitro Expansion and Characterization of Adult Human Brain Cells

1. In vitro expansion:
   1. Aspirate the culture medium and wash with phosphate-buffered saline (PBS), kept at room temperature, before adding 3 ml TrypLE.
   2. Incubate at 37 °C for 5-10 min until most of the cells detached. Gently tap the flasks to facilitate lifting of the cells; following addition of 3 volumes of growth medium, transfer the cells into a 15 ml conical tube and spin down at 157 x g for 5 min and resuspend the cells in the appropriate amount of growth medium.
   3. Passage the cells at a ratio of 1:3 for optimal yield into new T75 cell culture flasks.
      Note: We have passaged these cells until P5 without any signs of reduced reprogramming efficiency. While at P0 the culture contains a considerable fraction of endothelial cells (as assessed by CD34 expression), at subsequent passages their number becomes negligible.
2. Characterization of adult human brain cultures:
   1. Immunocytochemistry:
      1. For characterization of the cells, seed ~60,000 cells onto poly-D-lysine-coated glass cover slips in 500 μl fresh growth medium in 24-well tissue culture plates and incubate at 37 °C with 5% CO₂ and 5% O₂.
      2. For immunocytochemical analysis of the cultured human brain cells remove the medium and wash 3x with 1 ml of PBS.
      3. Fix cells by adding 500 μl of 4% paraformaldehyde in PBS for 15 min at room temperature.
      4. Transfer the glass cover slips into an appropriate humidified staining chamber and block with 80 μl PBS containing 3% bovine serum albumin (BSA) and 0.5% Triton X-100 for 1 hr at room temperature.
      5. Add primary antibodies diluted in the same solution (for dilution factors see table of specific reagents and equipment) and incubate overnight at 4 °C or 1 hr at room temperature.
         Note: A substantial but varying fraction of the cells at this stage of culturing are immunoreactive for platelet-derived growth factor receptor β (PDGFRβ), cluster of differentiation 146 (CD146), NG2 chondroitin sulfate proteoglycan, and smooth muscle actin (SMA), i.e. markers characteristic of microvessel-associated pericytes. Only a minority (<1%) express the astroglial markers glial fibrillary acidic protein (GFAP) and S100β. Furthermore, at this stage the culture should be devoid of any cells expressing the neuronal marker βIII-tubulin. Use multiple primary antibody combinations for co-labeling of different markers. Further confirmation of the expression of glial, neuronal, endothelial and pericyte markers can be obtained by reverse transcription and quantitative real time polymerase chain reaction (not described in this protocol).
      6. Following incubation with the primary antibodies wash three times with 1 ml PBS and add the corresponding secondary antibodies (in the appropriate dilutions) to the staining solution and incubate 1 hr at room temperature in the dark. Wash the cells 3x with PBS. If required, prior to mounting include staining with DAPI for 5 min at room temperature.
      7. Use antifading mounting medium and analyze the cover slips using an epifluorescence microscope or a confocal microscope.
   2. Flow cytometry:
      1. For analysis of surface marker expression using flow cytometry, grow cells to confluency in uncoated T25 or T75 cell culture flasks.
      2. Detach cells by using TrypLE as described before and resuspend in staining solution consisting of PBS + 0.5% BSA. Per staining reaction, resuspend 100,000-200,000 cells in 100 μl of staining solution. Prepare single staining reactions per antibody used (CD146-FITC, CD140b-PE, CD34-APC, CD13-FITC, respective isotype control antibodies).
      3. Add fluorochrome-conjugated antibodies to each staining solution (for dilutions see table of specific reagents and equipment) directly into the cell suspension and incubate at 4 °C for 30 min in the dark.
      4. Wash the cells three times with 500 μl PBS and spin down between each washing step at 157 x g for 5 min.
      5. Resuspend the cell pellet in 0.5-1 ml staining solution and transfer cells through a cell strainer (70 μm) to FACS tubes. Protect the sample from exposure to light.
      6. Vortex samples prior to placing them in the FACS instrument.
      7. To analyze the living cells and exclude debris and cell aggregates, gate for FSC-A and SSC-A. Cell duplets are specifically gated out by FSC-A and FSC-W. To determine the right gating conditions for the cell surface markers set the gates with isotype-matched antibody control conjugated to the respective fluorochrome. Then determine the proportion of antibody-bound cells.

3. Enrichment for Pericyte-derived Cells by Fluorescence Activated Cell Sorting

1. Purify the pericyte cell population (resuspended in growth medium) prior to transduction via FACS sorting using a 70 μm nozzle. Use CD34-APC in combination with CD140b-PE and CD146-FITC to select for pericytes. Sort for CD34-negative, CD140b-, and CD146-positive cells.
2. Collect the sorted cells in growth medium and plate onto poly-D-lysine-coated glass cover slips in 24-well tissue culture plates and incubate at 37 °C with 5% CO₂ and 5% O₂.
3. 48 h after sorting replace the medium with fresh growth medium and perform transduction as described in  Protocol 4.

4. Retroviral Transduction of Pericyte-derived Cells and Reprogramming into Induced Neurons

Note: Refer to local biosafety guidelines when handling retroviral particles. In Germany the use of the retroviruses employed here require biosafety level 2. For production of retroviral particles refer to protocol⁶. For retroviral constructs used for reprogramming, refer to Karow et al. (2012). Retroviruses were pseudotyped with VSV-G (vesicular stomatitis virus-glycoprotein)⁵. For production please refer to Gavrilescu et al⁶.

1. 24 hr prior to retroviral transduction seed ~60,000 cells onto poly-D-lysine-coated glass cover slips in 500 μl fresh growth medium in 24-well tissue culture plates and incubate at 37 °C with 5% CO₂ and 5% O₂.
2. The next day, replace the growth medium with 500 μl fresh, prewarmed growth medium and add 1 μl of the respective concentrated supernatants containing retroviral particles (pCAG-IRES-dsred for control, especially during establishing the protocol in the lab), pCAG-ascl1-IRES-dsred, pCAG-sox2-IRES-gfp.
3. One day later, remove the medium including the viral particles from the cells and add 1 ml fresh, prewarmed B27-differentiation medium, composed of 49 ml DMEM high glucose with GlutaMAX and 1 ml B27 serum-free supplement. Maintain the cells in culture at 37 °C in 5% CO₂ and 5% O₂ for 4-8 weeks without changing the medium as repetitive medium changes become harmful as iNs mature.

5. Characterization of Induced Neurons by Immunocytochemistry

1. To determine the cellular identity of the transduced cells, in particular to demonstrate the acquisition of a neuronal phenotype, perform immunocytochemistry against neuronal antigens such as bIII-tubulin, MAP2, and NeuN (for antibodies and their respective dilutions, see table of specific reagents and equipment; follow the same procedure as indicated in 2.2.1).
2. For improving the maturation of PdiNs, co-culture these cells with neurons derived from E14 mouse cerebral cortices. To this end, dissect the cerebral cortex and dissociate the tissue mechanically with a fire-polished glass Pasteur pipette. Add 10,000-50,000 murine neurons to cultures of pericyte-derived cells 2-3 weeks following transduction with sox2- and ascl1-encoding retroviruses and continue culturing. Transduced cells can be distinguished from murine neurons by their reporter (GFP and dsRed) expression, either by live epifluorescence or following immunocytochemistry for GFP and dsRed.
3. To assess the formation of synapses onto PdiNs by murine neurons, perform immunocytochemistry against vesicular neurotransmitter receptors such as vesicular glutamate transporter 1 (vGluT1).
4. Probe transduced cells for their electrical properties (i.e. ability of generating action potentials) and the establishment of functional synapses by patch-clamp electrophysiology. For details of the procedure see Heinrich et al. (2011).

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