Media | Reagent | Amount to mix | Final concentration (%) |
Fibroblast media | DMEM, high glucose, GlutaMAX | 500 mL | 89 |
Fetal Bovine Serum | 50 mL | 10 | |
Antibiotic-Antimycotic | 5 mL | 1 | |
Base media | DMEM/F12 + Glutamax | 500 mL | 97 |
N-2 | 5 mL | 1 | |
B-27 | 5 mL | 1 | |
Antibiotic-Antimycotic | 5 mL | 1 | |
Conversion media | Base media | 50 mL | 99.9 |
FGF | 1 µL | 0.02 (20 ng/mL) | |
EGF | 1 µL | 0.02 (20 ng/mL) | |
Heparin | 50 µL | 0.1 (5 µg/mL) | |
Neural Progenitor Cell (NPC) Media | DMEM/F12 + Glutamax | 500 mL | 96.9 |
N-2 | 5 mL | 1 | |
B-27 | 5 mL | 1 | |
Antibiotic-Antimycotic | 5 mL | 1 | |
FGF | 10 uL | 0.002 | |
Astrocyte Media | DMEM, high glucose, GlutaMAX | 500 mL | 89 |
Fetal Bovine Serum | 50 mL | 10 | |
Antibiotic-Antimycotic | 5 mL | 1 | |
N-2 | 1 mL | 0.2 | |
Media must be filtered after mixing all components. Conversion media (with factors) must be prepared fresh every week. |
Table 1. Media recipes for all cell types included in the protocol. Media should be filtered after mixing all components with either a sterile vacuum filtration system for big volumes or syringe and 0.2 um syringe filters. Conversion media (with factors) must be prepared fresh every week.
1. Direct conversion of adult human fibroblasts to neuronal progenitor cells
NOTE: A schematic timeline of this protocol can be reviewed in Meyer (2014)19.
2. Conversion and iNPC splitting procedure
3. Generating induced astrocytes from NPCs
Plate Type | Astrocytes per Well |
384-well | 2500 |
96-well | 10000 |
24-well | 40000 |
6-well | 150000 |
Table 2. Recommended seeding density of iAs per plate area. Typical number of iAs seeded to generate a monolayer on the most common tissue culture plates.
4. Preparing and defrosting portions for astrocyte differentiation from frozen NPC stocks (alternative to step 3)
NOTE: As an alternative to maintaining iNPCs in culture, astrocytes can also be produced directly from a frozen stock. To do so, the iNPCs are frozen in smaller portions. Table 3 shows suggested freezing and thawing volumes for portions to be defrosted into a 10 cm dish containing 10 mL of Astrocyte media.
Portion Size | Cell Suspension (µL) | Freezing media (µL) | Total volume (µL) | Defrosts Into |
4x | 400 | 400 | 800 | Two 10-cm dishes |
2x | 200 | 200 | 400 | One 10-cm dish |
Table 3. Instructions on how to portion iNPC to generate iAs. Proportion of iNPC suspension and freezing media to generate portions. Note that the final DMSO concentration is 10% when cell suspension is mixed with freezing media.
This protocol allows the rapid and easy generation of iNPCs directly from human skin fibroblasts using retroviruses containing the Yamanaka factors. This method allows bypassing the stem cell state and the need for clonal selection, thereby avoiding clonal variation. Important steps to keep in mind while cells are undergoing the conversion process are the fibroblast seeding density, media pH and keeping the cells at an optimal confluency. Examples of optimal splitting confluency and morphology changes during the conversion process can be found in Figure 1.
Figure 1. Representative images of the conversion process after adding the retroviral mix of Yamanaka factors. Fibroblasts were seeded and transduced twenty-four hours later with Yamanaka factors. Two days post virus (DPV), media was changed to conversion media. Cells were cultured in conversion media until they were ready to passage (13 DPV). Cells were seeded post passage to reach 80% confluence the following day (14 DPV). Subsequent passages maintained this density until neuronal progenitor cells begin rapidly dividing. Scale bar is 200 µm. Please click here to view a larger version of this figure.
After the conversion process is complete, the NPCs will show strongly reduced expression or no expression of fibroblast markers and morphology, and express NPC specific cell markers (Figure 2). Moreover, they can also be used for generating different cell lines, like iNs, iOs, and iAs.
Figure 2. Cells that undergo the conversion process express cell-specific markers. Patient fibroblasts (A), induced Neuronal Progenitor Cells (iNPCs (B) and induced Astrocytes (iAs (C) cells were seeded on glass coverslips in a 24 well tissue culture treated plate. Cells were immunostained for: fibroblast specific cell markers (A), Vimentin (green) and TE7 (red), iNPC specific cell marker (B), Nestin (red), and iAs cell marker (C) GFAP (purple) and iNPC marker Nestin (green). Scale bar is 50 µm. Please click here to view a larger version of this figure.
In our experience, iAs in particular are very valuable for drug and disease mechanism testing as they can be generated in pure populations (98% or more GFAP positive cells29) at reproducible large numbers. iAs can be differentiated from iNPCs by taking a small aliquot of cells during splitting or a previously frozen iNPC portion and directly plating in astrocyte media. Important considerations during this process are maintaining the iNPCs initial seeding density low (Figure 3 and Figure 4), as a high density has been shown to hinder the differentiation process (Figure 4) and paying additional attention to the media pH, as acidification can activate even healthy astrocytes.
Figure 3. Representative images of the iAs generation process from iNPCs. iNPCs are seeded in Astrocyte media (Table 1) at a low seeding density. A good seeding density is about 10% on the first day post seeding (DPS) (left); however, this density can be adjusted according to the growth rate of cells. Typical iAs morphology can be observed after 5 DPS (middle). In some cases, aberrant astrocyte morphology can be observed, with long, spiky extensions. This change is indicative of astrocyte activation and can be secondary to disease state or incorrect culturing techniques (right). Scale bar is 200 µm. Please click here to view a larger version of this figure.
Figure 4. iNPC seeding density affects the iAs differentiation efficiency. Control iNPC lines were seeded at low (A) and high (B) density in Astrocyte media to demonstrate effects of seeding density on differentiation. 5 DPS, the low-density line expressed astrocyte-specific markers, while the high-density line shows a mixture of iNPC and iAs markers with a marked iNPC-like morphology. Scale bar is 50 µm. Please click here to view a larger version of this figure.
Supplemental Figure 1. Additional figures of the conversion process after adding the retroviral mix of Yamanaka factors. Images of the conversion process at 12 DPV (1 day before passage) and 19 DPV (6 days after passage). Note the difference on morphology between cells before and after passage, at 12 DPV cells have a ball-like structure morphology. After a passage and several days on conversion media (table 1) cells start displaying a NPC-like morphology. Scale bar is 200 µm. Please click here to download this File.
100mm x 2mm Style dish, Cell culture treated, Nonpyrogenic | Corning | 430167 | Tissue culture |
15 ml conical screw cap centrifuge tubes, copolymer polypropylene | USA Scientific | 1475-1611 | Used for lifting and centrifuge cells |
50mL Conical Centrifuge Tubes | USA Scientific | 1500-1811 | Media preparation |
Antibiotic-Antimycotic (100X) | Gibco | 15240062 | Antibiotic with antifungal activity for media preparation |
B-27 Supplement (50X), serum free | Invitrogen | 17504044 | For NPC and base media |
Cryogenic vials 1.2ML | Corning | CLS430658-500EA | For freezing cell stocks |
DMEM, high glucose, GlutaMAX Supplement, pyruvate | Gibco | 10569010 | For fibroblast and Astrocyte media |
DMEM/F-12, GlutaMAX supplement | Gibco | 10565042 | For NPC and base media |
DMSO | Sigma | D2438-50ML | For freezing cell stocks |
Dulbecco’s Phosphate Buffered Saline (PBS) | Gibco | 14190136 | Referred in protocol as PBS. For fibronectin dilution and cell wash |
EZ Retrovirus iPSC Reprogramming Kit | ALSTEM | RF101 | Retrovirus containing the Yamanaka factors. Virus can also be made in-house. |
Fetal Bovine Serum, certified | Gibco | 16000-036 | Referred in protocol as FBS . For Fibroblast and Astrocyte media |
Fisherbrand Sterile Syringes for Single Use | Fisher | 14-955-461 | Filter media (50mL) |
Heparin sodium salt from porcine intestinal mucosa | Sigma | H3149-10KU | Referred in protocol as Heparin. Used in conversion media. Powder diluted in ultrapure water. Final stock concentration of 5000X |
Human Plasma Fibronectin Purified Protein | Millipore Sigma | FC010-10MG | Referred in protocol as Fibronectin, used in 1:200 dilution for coating. |
Isopropanol (technical grade) | Fisher Scientific | S25372A | For freezing cell stocks |
Mr. Frosty | Thermo Fisher | 5100-0001 | For freezing cell stocks |
N-2 Supplement (100X) | Gibco | 17502048 | For Astrocyte, NPC and base media |
Recombinant Human EGF | Preprotech | AF-100-15 | Referred in protocol as EGF, used in conversion media. Powder diluted in PBS, final concentration of 1mg/mL, stored in small frozen aliquots |
Recombinant Human FGF-basic | Preprotech | 100-18B | Referred in protocol as FGF, used in NPC and conversion media. Powder diluted in PBS, final concentration of 1mg/mL, stored in small frozen aliquots |
StemPro Accutase Cell Dissociation Reagent | Gibco | A1110501 | Referred in protocol as Accutase. Used for lifting during the conversion process and NPCs. |
Stericup Quick Release-GP Sterile Vacuum Filtration System | Millipore Sigma | S2GPU05RE | Media filtration |
Tissue culture plate, 6 well, Flat bottom with Low evaporation lid | Fisher | 08-772-1B | Tissue culture |
Trypsin-EDTA (0.05%), phenol red | Invitrogen | 25300062 | Lifting of Fibroblasts and Astrocytes |
Whatman Puradisc 25 syringe filters, 0.2 μm, PES sterile | Millipore Sigma | 80-2502 | Filter media (50mL) |
Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.
Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.
Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.