generating neurons by reprogramming human dermal fibroblast cells

Generating Neurons by Reprogramming Human Dermal Fibroblast Cells

Prepare a cellular suspension of 1,320,000 cells in 13.2 milliliters of fibroblast medium in order to achieve 100,000 cells per milliliter of medium. After aspirating the gelatin from the plate, wash the plate with Dulbecco&#39;s phosphate-buffered saline twice. Then, add 500 microliters of cell suspension to each of the wells, and incubate at 37 degrees Celsius overnight.
Warm 13.2 milliliters of fibroblast medium to 37 degrees Celsius. Then, thaw the lentiviral vector in the laminar hood at room temperature. Add the required volume of lentivirus necessary to infect the adult human dermal fibroblasts at a multiplicity of infection of 20 to the medium without any transduction enhancers.
Then, replace the medium with 500 microliters of fibroblast medium with the addition of the lentiviral vector. Incubate the plate at 37 degrees Celsius overnight. The following day, replace the medium containing lentivirus with fresh fibroblast medium without the lentiviral vector. On the third day after viral transduction, remove the fibroblast medium and add 500 microliters of early neuronal conversion medium.
Twice or thrice a week, replace 225 microliters of old medium from each well with 250 microliters of fresh, early neuronal conversion medium. On the 18th day after viral transduction, remove the early neuronal conversion medium and replace with 500 microliters of late neuronal conversion medium. Keep changing the medium every two to three days as before until the 25th day or the experimental endpoint.

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1. Preparation of Skin Fibroblasts for Reprogramming
<ol>
	<li>Using an automated cell thawing system or a 37 &#176;C water bath, thaw the adult human dermal fibroblasts (aHDFs) and plate 200,000 per T75 flask (count with an automated cell counter) in 10 mL of fibroblast medium (see&#160;Table 1) at 37 &#176;C in 5% CO2.</li>
	<li>Perform a complete medium change with fibroblast medium on the next day.</li>
	<li>Change the fibroblast medium every 3&#8211;4 days until the cells reach 95% confluency. 
	NOTE: One confluent flask will contain approximately 1,000,000 cells.</li>
</ol>
2. Plating for Reprogramming (Day &#8722;1)
NOTE: It is recommended to use a gelatin coating for short term experiments (up to 30 days); alternatively, for long term experiments it is recommended to start on a poly-L-ornithine, fibronectin and laminin (PFL) coating.
<ol>
	<li>60 min before plating the aHDFs for reprogramming, coat a 24-well plate with 0.1% gelatin (250 &#181;L/well) and incubate at 37 &#176;C.</li>
	<li>Aspirate the fibroblast medium on the aHDFs. Wash once with Dulbecco&#39;s phosphate buffer saline (DPBS). Dissociate the cells with 0.05% trypsin (1.5 mL per T75 flask) at 37 &#176;C for 3&#8211;5 min.</li>
	<li>Add fibroblast medium to neutralize the trypsin (3 mL per flush per T75 flask) and collect the detached cells in a 15 mL tube by flushing out the cells in the flask twice.</li>
	<li>Spin down the cells at 400 x g for 5 min. Discard supernatant and resuspend the cell pellet in 1 mL of fibroblast medium.</li>
	<li>Count the cells using an automated cell counter (to ensure a good quality conversion check that the cell viability is above 90% with trypan blue staining).</li>
	<li>For a complete 24-well plate, prepare a suspension of 1,320,000 cells in 13.2 mL of fibroblast medium to achieve a suspension of 100,000 cells/mL of medium (or 55,000 cells/well in 550 &#181;L of fibroblast medium multiplied by the number of wells needed).</li>
	<li>Aspirate the gelatin from the plate and wash twice with DPBS. Add 500 &#181;L of the cell suspension to each well and incubate overnight at 37 &#176;C in 5% CO2.</li>
</ol>
3. Viral Transduction (Day 0)
NOTE: Working with lentiviral particles requires category 2 equipment and the use of an agent to neutralize the virus. Wearing double pairs of gloves is also strongly recommended.
<ol>
	<li>Warm up 13.2 mL of fibroblast medium to 37 &#176;C.</li>
	<li>Thaw a lentiviral vector containing the transcription factors&#160;achaete-scute family bHLH transcription factor 1 (Ascl1)&#160;and&#160;Brn2&#160;with two short hairpin RNAs (shRNA) targeting RE1-silencing transcription factor (REST) at room temperature.</li>
	<li>Add the necessary volume of lentivirus to infect the aHDFs at multiplicity of infection (MOI) of 20 to the medium without any transduction enhancers. 
	<img alt="Equation 1" src="/files/ftp_upload/22414/22414_equation1.jpg" style="margin: auto;" /></li>
	<li>Replace the medium in the 24-well plate with fibroblast medium containing the lentiviral vector (500 &#181;L/well) and incubate overnight at 37 &#176;C in 5% CO2.</li>
	<li>The next day, replace the medium in the wells with fresh fibroblast medium without the lentiviral vector. 
	NOTE: The medium is considered infectious for 7 days and as such, adequate protection and handling procedures should be used during the first week following viral transduction.</li>
</ol>
4. Maintenance of the Converting Cells
NOTE: Once conversion begins cells are susceptible to lifting; take care to tip the plate up and use a 1,000 &#181;L pipette when removing media to avoid cells detaching.
<ol>
	<li>On day 3, remove the fibroblast medium and add 500 &#181;L of early neuronal conversion medium (see&#160;Table 1).</li>
	<li>Two to three times a week, take out 225 &#181;L of old medium from the well and add-in 250 &#181;L of fresh early neuronal conversion medium.</li>
	<li>On day 18, remove all of the medium from each well and replace with 500 &#181;L of late neuronal conversion medium (see&#160;Table 1).</li>
	<li>Continue to change half of the medium as above with late neuronal conversion medium every 2&#8211;3 days until day 25 or experiment endpoint (Figure 1A).</li>
</ol>
Table 1. &#160;Composition of the different media used.&#160;Full description of the composition for media needed in this protocol including fibroblast medium, early neuronal conversion medium, late neuronal conversion medium.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Stock Concentration</td>
			<td>Working Concentration</td>
		</tr>
		<tr>
			<td colspan="3">Fibroblast medium</td>
		</tr>
		<tr>
			<td>Basal medium</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>10,000 U/mL</td>
			<td>100 mg/mL</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>N/A</td>
			<td>10%</td>
		</tr>
		<tr>
			<td colspan="2">Early neuronal conversion medium (ENM)</td>
			<td></td>
		</tr>
		<tr>
			<td>Neural differentiation medium&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>10,000 U/mL</td>
			<td>100 mg/mL</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>10 mM</td>
			<td>2 &#181;M</td>
		</tr>
		<tr>
			<td>SB-431542</td>
			<td>20 mM</td>
			<td>10 &#181;M</td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>100 &#181;g/mL</td>
			<td>0.5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>LDN-1931189</td>
			<td>10 mM</td>
			<td>0.5 &#181;M</td>
		</tr>
		<tr>
			<td>VPA</td>
			<td>1 M</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>LM-22A4</td>
			<td>20 mM</td>
			<td>2 &#181;M</td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>20 &#181;g/mL</td>
			<td>2 ng/mL</td>
		</tr>
		<tr>
			<td>NT3</td>
			<td>10 &#181;g/mL</td>
			<td>10 ng/&#181;L</td>
		</tr>
		<tr>
			<td>db-cAMP</td>
			<td>50 mM</td>
			<td>0.5 mM</td>
		</tr>
		<tr>
			<td colspan="2">Late neuronal conversion medium (LNM)</td>
			<td></td>
		</tr>
		<tr>
			<td>Neural differentiation medium&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>10,000 U/mL</td>
			<td>100 mg/mL</td>
		</tr>
		<tr>
			<td>LM-22A4</td>
			<td>20 mM</td>
			<td>2 &#181;M</td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>20 &#181;g/mL</td>
			<td>2 ng/mL</td>
		</tr>
		<tr>
			<td>NT3</td>
			<td>10 &#181;g/mL</td>
			<td>10 ng/&#181;L</td>
		</tr>
		<tr>
			<td>db-cAMP</td>
			<td>50 mM</td>
			<td>0.5 mM</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell Lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adult human dermal fibroblasts</td>
			<td></td>
			<td></td>
			<td>[C2 passage #7] Donor was a 67 year old female. Cells obtained from the Parkinson&#8217;s Disease Research and Huntington&#8217;s disease clinics at the John van Geest Centre for Brain Repair (Cambridge, UK).</td>
		</tr>
		<tr>
			<td>Reagents for Fibroblast Culture, Transduction and Conversion</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline (DPBS) [-CaCl2, -MgCl2]</td>
			<td>Gibco</td>
			<td>14190094</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA [0.5%]</td>
			<td>Gibco</td>
			<td>15400-054</td>
			<td>Dilute to 0.05% in DPBS.</td>
		</tr>
		<tr>
			<td>Virkon (agent used to neutralize virus)</td>
			<td>Viroderm</td>
			<td>7511</td>
			<td>Dilute to 1% solution with warm water.</td>
		</tr>
		<tr>
			<td>Milli-Q Water</td>
			<td>Millipore</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Basal medium - Dulbecco&#8217;s Modified Eagle Medium (DMEM) + GlutaMax</td>
			<td>Gibco</td>
			<td>61965059</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin [10,000 U/ mL]</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>CryoMACS&#174; dimethyl sulfoxide (DMSO) 10</td>
			<td>Miltenyi</td>
			<td>170-076-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Neural differentiation medium - NDiff 227</td>
			<td>Takara-Clontech</td>
			<td>Y40002</td>
			<td></td>
		</tr>
		<tr>
			<td>LM-22A4</td>
			<td>Tocris</td>
			<td>4607</td>
			<td>Dilute 10 mg in 1450 &#181;L DMSO. 
			Stock concentration: 20 mM.</td>
		</tr>
		<tr>
			<td>Glial cell line-derived neurotrophic factor (GDNF) [recombinant human]</td>
			<td>R&#38;D systems</td>
			<td>212-GD-010</td>
			<td>Dilute 10 ug in 500 &#181;L 0,1% BSA in DPBS. Stock concentration: 20 &#181;g/mL.</td>
		</tr>
		<tr>
			<td>NT3 [recombinant human]</td>
			<td>R&#38;D systems</td>
			<td>267-N3-025</td>
			<td>Dilute 25 &#181;g in 2,5 mL 0,1% BSA in DPBS. Stock concentration: 10 &#181;g/mL.</td>
		</tr>
		<tr>
			<td>db-cAMP</td>
			<td>Sigma Aldrich</td>
			<td>D0627</td>
			<td>Dilute 1 g in 40,7 mL Milli-Q water. 
			Filter and make 500 &#181;L aliquots or stock tubes of 10 mL. Stock concentration: 50 mM.</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Axon</td>
			<td>1386</td>
			<td>Dilute 2 &#181;g in 429,8 &#181;L DMSO. 
			Stock concentration: 10 mM.</td>
		</tr>
		<tr>
			<td>LDN-193189</td>
			<td>Axon</td>
			<td>1509</td>
			<td>Dilute 2 mg in 360 &#181;L DMSO. 
			Stock concentration: 10 mM.</td>
		</tr>
		<tr>
			<td>Valproic acid sodium salt (VPA)</td>
			<td>Merck Millipore</td>
			<td>676380</td>
			<td>Dilute 5 g in Milli-Q water to acheive a stock concentration of 1 M. CAUTION: Avoid ingestion, contact with skin, and breathing dust formation.</td>
		</tr>
		<tr>
			<td>Reagents for Coatings</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma Aldrich</td>
			<td>G2500</td>
			<td>Dilute to 0.1% in Milli-Q water.</td>
		</tr>
		<tr>
			<td>Poly-L-ornithine</td>
			<td>Sigma Aldrich</td>
			<td>P3655</td>
			<td>Dissolve in Milli-Q water. Use at 15&#181;g/mL.</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>ThermoFisher Scientific</td>
			<td>33010-018</td>
			<td>2 mL of Milli-Q water + 70 &#181;L 0,25 M NaOH. Use at 5 &#181;g/mL.</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>ThermoFisher Scientific</td>
			<td>23017-015</td>
			<td>Store at -80&#176;C. Thaw on ice, keep cool and aliquot 30 &#181;L. Use at 5 &#181;g/mL.</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T75 flask [Nunclon Delta Surface]</td>
			<td>ThermoFisher Scientific</td>
			<td>156499</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate [Nunc]</td>
			<td>ThermoFisher Scientific</td>
			<td>142485</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL polypropylene tube</td>
			<td>Sigma Aldrich</td>
			<td>Z336769</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL falcon tube</td>
			<td>Sarstedt</td>
			<td>6,25,54,502</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL falcon tube</td>
			<td>Sarstedt</td>
			<td>6,25,47,254</td>
			<td></td>
		</tr>
		<tr>
			<td>Pippette controller</td>
			<td></td>
			<td></td>
			<td>For pipetting volumes 1-25 mL.</td>
		</tr>
		<tr>
			<td>Sterile serological pipettes: 5, 10 and 25 mL</td>
			<td>Sarstedt</td>
			<td>86.1253.001, 86.1254.001, 86.1685.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Adjustable volume pipettors: 5, 20, 200, and 1,000 &#181;L</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile pipette tips</td>
			<td></td>
			<td></td>
			<td>For pipetting volumes of 0.5 - 1,000 &#181;L.</td>
		</tr>
		<tr>
			<td>ThawSTAR Automated Cell Thawing System</td>
			<td>BioCision</td>
			<td>BCS-601</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II Automated Cell Counter</td>
			<td>ThermoFisher Scientific</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counting chambers [50 slides] and trypan blue [0.4%]</td>
			<td>ThermoFisher Scientific</td>
			<td>C10228</td>
			<td>For use with Countess II Automated Cell Counter.</td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Humidified 5% CO2 37 &#176;C incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td>Suitable for 1,5, 15 and 50 mL tubes.</td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted fluorescence microscope</td>
			<td>Leica</td>
			<td>DMI6000 B</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Shrigley, S., et al. <a href="https://app.jove.com/v/56904">Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts.</a> J. Vis. Exp. (2018)
The video demonstrates the generation of neurons from adult human dermal fibroblasts (aHDFs). The aHDFs are reprogrammed using a lentiviral vector carrying genes for neuronal transcription factors and short hairpin RNAs (shRNAs). The shRNAs inhibit the expression of a neuronal transcriptional repressor, and the neuronal transcription factors activate genes to differentiate aHDFs into neurons. These reprogrammed aHDFs are cultured in an early and a late neuronal conversion medium to generate mature neurons.

<img alt="Figure 1" src="/files/ftp_upload/22414/22414_Figure1.jpg" /> 
Figure 1: Evolution of the iN conversion over time.&#160;(A) Timeline of the experiment and map of the construct packaged in a lentivirus used to reprogram the adult human dermal fibroblasts. Each black arrow represents a medium change. (B) Representative phase contrast images depicting the changes in morphology of cells during the conversion process between day ...

1. Preparation of Skin Fibroblasts for Reprogramming

	Using an automated cell thawing system or a 37 &#176;C water bath, thaw the adult human dermal ...

Introduce adult human dermal fibroblasts or aHDFs, suspended in a fibroblast medium, into an extracellular matrix protein-coated microplate. Incubate to allow cell adherence.
Introduce a transgenic lentiviral vector. Upon internalization into the host cell, the viral RNA is reverse-transcribed into DNA, which integrates into the host genome.
The viral DNA expresses transcription factors and short hairpin RNAs, or shRNAs. Cellular nucleases process the shRNAs into small interfering RNAs or siRNAs.
The generated siRNAs degrade the mRNA encoding transcriptional repressors, thereby inhibiting their expression and enabling the activation of neuronal differentiation genes.
The transcription factors activate the genes, promoting neuronal differentiation of the aHDFs.
Remove the medium containing the non-internalized vectors.
Add an early neuronal conversion medium containing growth factors and small molecules that induce differentiation into neuron-like cells.
Replace the medium with a late neuronal conversion medium containing neurotrophic factors for maturation into neurons.
The mature neurons are ready for downstream assays.

21 ビュー. ヒト真皮線維芽細胞のリプログラミングによるニューロンの生成

ビデオ: ヒト真皮線維芽細胞のリプログラミングによるニューロンの生成 - 実験

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

転写因子の強制発現によるヒト線維芽細胞のヘモジェニックリプログラミング

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

JoVE Journal

発生生物学

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

多能性幹細胞からのヒト神経細胞および希突起膠細胞の作製によるニューロン-希突起膠細胞相互作用のモデル化

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

神経科学

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

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.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

線維芽細胞からニューロン前駆細胞への直接変換とアストロサイトへの分化を用いた神経疾患のインビトロモデリング

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...

Generating Neurons by Reprogramming Human Dermal Fibroblast Cells

記録

Generating Neurons by Reprogramming Human Dermal Fibroblast Cells