Name: direct reprogramming of mouse fibroblasts into melanocytes
Uploaded: 2021-08-27T14:30:00
Description: Watch this Scientific Journal Video about Direct Reprogramming of Mouse Fibroblasts into Melanocytes at JoVE.com

This study was partially supported by grants from the National Natural Science Foundation of China (82070638 and 81770621) and the Natural Science Foundation of Jiangsu Province (BK20180281).

This work was approved by the Laboratory Animal Management and Use Committee at Jiangsu University (UJS-IACUC-AP--20190305010). The experiments were performed in strict accordance with the standards established by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). There were no experiments involving humans, so this work did not need approval from the human research ethics committee. Refer to the Table of Materials for details about reagents.</...

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	<li>Ezzedine, K., Eleftheriadou, V., Whitton, M., van Geel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitiligo.">Vitiligo.</a> Lancet. 386 (9988), 74-84 (2015).</li><li>Picardo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitiligo.">Vitiligo.</a> Nature Reviews. Disease Primers. 1, 15011 (2015).</li><li>Speeckaert, R., van Geel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitiligo:+An+update+on+pathophysiology+and+treatment+options.">Vitiligo: An update on pathophysiology and treatment options.</a> American Journal of Clinical Dermatology. 18 (6), 733-744 (2017).</li><li>Cortelazzi, C., Pellacani, G., Raposio, E., Di Nuzzo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitiligo+management:+combination+of+surgical+treatment+and+phototherapy+under+reflectance+confocal+microscopy+monitoring.">Vitiligo management: combination of surgical treatment and phototherapy under reflectance confocal microscopy monitoring.</a> European Review for Medical and Pharmacological Sciences. 24 (13), 7366-7371 (2020).</li><li>Mohammad, T. F., Hamzavi, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+therapies+for+vitiligo.">Surgical therapies for vitiligo.</a> Dermatologic Clinics. 35 (2), 193-203 (2017).</li><li>Bishnoi, A., Parsad, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+molecular+aspects+of+vitiligo+treatments.">Clinical and molecular aspects of vitiligo treatments.</a> International journal of molecular sciences. 19 (5), 1509 (2018).</li><li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+decade+of+transcription+factor-mediated+reprogramming+to+pluripotency.">A decade of transcription factor-mediated reprogramming to pluripotency.</a> Nature Reviews. Molecular Cell Biology. 17 (3), 183-193 (2016).</li><li>Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+stem+cell-based+cell+therapy-promise+and+challenges.">Pluripotent stem cell-based cell therapy-promise and challenges.</a> Cell Stem Cell. 27 (4), 523-531 (2020).</li><li>Xu, J., Du, Y., Deng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+lineage+reprogramming:+strategies,+mechanisms,+and+applications.">Direct lineage reprogramming: strategies, mechanisms, and applications.</a> Cell Stem Cell. 16 (2), 119-134 (2015).</li><li>Ieda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+reprogramming+of+fibroblasts+into+functional+cardiomyocytes+by+defined+factors.">Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors.</a> Cell. 142 (3), 375-386 (2010).</li><li>Gasc&#243;n, S., Masserdotti, G., Russo, G. L., G&#246;tz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+neuronal+reprogramming:+achievements,+hurdles,+and+new+roads+to+success.">Direct neuronal reprogramming: achievements, hurdles, and new roads to success.</a> Cell Stem Cell. 21 (1), 18-34 (2017).</li><li>Atkinson, P. J., Kim, G. S., Cheng, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+cellular+reprogramming+and+inner+ear+regeneration.">Direct cellular reprogramming and inner ear regeneration.</a> Expert Opinion on Biological Therapy. 19 (2), 129-139 (2019).</li><li>Kurita, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+reprogramming+of+wound-resident+cells+generates+skin+epithelial+tissue.">In vivo reprogramming of wound-resident cells generates skin epithelial tissue.</a> Nature. 561 (7722), 243-247 (2018).</li><li>Yang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+conversion+of+mouse+and+human+fibroblasts+to+functional+melanocytes+by+defined+factors.">Direct conversion of mouse and human fibroblasts to functional melanocytes by defined factors.</a> Nature Communications. 5, 5807 (2014).</li><li>Fehrenbach, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+tumorigenic+potential+upon+transdifferentiation+from+keratinocytic+into+melanocytic+lineage.">Loss of tumorigenic potential upon transdifferentiation from keratinocytic into melanocytic lineage.</a> Scientific Reports. 6, 28891 (2016).</li><li>Majumdar, G., Vera, S., Elam, M. B., Raghow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+streamlined+protocol+for+extracting+RNA+and+genomic+DNA+from+archived+human+blood+and+muscle.">A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle.</a> Analytical Biochemistry. 474, 25-27 (2015).</li><li>Bachman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse-transcription+PCR+(RT-PCR).">Reverse-transcription PCR (RT-PCR).</a> Methods in Enzymology. 530, 67-74 (2013).</li><li>Donaldson, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+staining.">Immunofluorescence staining.</a> Current Protocols in Cell Biology. 69 (1), 1-7 (2015).</li><li>Yin, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-viral+vectors+for+gene-based+therapy.">Non-viral vectors for gene-based therapy.</a> Nature Reviews. Genetics. 15 (8), 541-555 (2014).</li></ol>

The quality of the virus is crucial for the success of direct reprogramming to melanocytes in this protocol. The method of packaging and concentrating viruses in this protocol is simple and easy to repeat and does not rely on any other auxiliary concentrated reagent. This protocol can be followed successfully in most laboratories. To ensure the quality of the concentrated virus, the following points need special attention. One is the cell status of HEK-293T. Although HEK-293T cells are immortalized cells, the cells used ...

Vitiligo is a skin disease that seriously affects the physical and mental health of the affected individuals. For various reasons, including metabolic abnormalities, oxidative stress, generation of inflammatory mediators, cell detachment, and autoimmune response, the functional melanocytes are lost, and the secretion of melanin is stopped, leading to the development of vitiligo1,2. This condition occurs widely and is particularly problematic on the face. The main treatment is the systemic use of corticosteroids and immunomodulators. Phototherapy can be used for systemic or local diseases, and there are surgical treatments, such as perforated skin transplantation and autologous melanocyte transplantation3,4,5. However, patients who use drug therapy and phototherapy are prone to relapse, and these treatments have poor long-term therapeutic effects. Surgical treatment is traumatic and only moderately effective2,6. Therefore, a new and effective therapeutic strategy is needed for vitiligo.
The reprogramming of induced pluripotent stem cells (iPSCs) reverses these cells from their terminal state to a pluripotent state, a process mediated by the transcription factors, Oct4, Sox2, Klf4, and c-Myc7. However, due to the possibility of tumorigenicity and the long production time, this technology has been met with skepticism when applied to clinical settings8. Direct reprogramming is a technology that makes one type of a terminal cell transform into another type of a terminal cell9. This process is achieved by suitable transcription factors. Various cells have already been directly reprogrammed successfully, including cardiomyocytes10, neurons11, and cochlear hair cells12. Some researchers have even reprogrammed skin tissue directly in situ, which can be used for wound repair13. The advantages of direct reprogramming include reduced wait times and costs, lower risk of cancer, fewer ethical problems, and a better understanding of the mechanism underlying cell fate determination9.
Although the direct reprogramming method is widely used, there is currently no definite method for the direct reprogramming of skin cells into melanocytes, especially because of the numerous transcription factors to be considered14,15. The transcription factors, Mitf, Sox10, and Pax3, have been used for direct reprogramming of skin cells into melanocytes14. In contrast, the combination of MITF, PAX3, SOX2, and SOX9 has also been used for direct reprogramming of skin cells into human melanocytes in another study15. In this protocol, despite the use of a different screening method, the same result was obtained with the combination of Mitf, Sox10, and Pax3 for direct reprogramming of skin cells into melanocytes as described previously14. Developing a system to generate melanocytes from other skin cells can provide a scheme for transforming other skin cells of vitiligo patients into melanocytes. Hence, it is crucial to construct a simple and efficient method for this direct reprogramming to generate melanocytes successfully.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300-062</td>
			<td>Stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>0.45 &#956;M filter</td>
			<td>Millipore</td>
			<td>SLHVR33RB</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL polystyrene round bottom tube</td>
			<td>Falcon</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr>
			<td>95%/100% ethanol</td>
			<td>LANBAO</td>
			<td>210106</td>
			<td>Stored at RT</td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>Sigma</td>
			<td>A2786</td>
			<td>Stock concentration 40 mg/mL 
			Final concentration 24 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 Goat anti-Mouse IgG2a</td>
			<td>Invitrogen</td>
			<td>A21137</td>
			<td>Dilution of 1:500 to use</td>
		</tr>
		<tr>
			<td>Antibiotics(Pen/Strep)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Anti-TRP1/TYRP1 Antibody</td>
			<td>Millipore</td>
			<td>MABC592</td>
			<td>Host/Isotype: Mouse IgG2a 
			Species reactivity: Mouse/Human 
			Dilution of 1:200 to use</td>
		</tr>
		<tr>
			<td>Anti-TRP2/DCT Antibody</td>
			<td>Abcam</td>
			<td>ab74073</td>
			<td>Host/Isotype: Rabbit IgG 
			Species reactivity: Mouse/Human Dilution of 1:200 to use</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Stemgent</td>
			<td>04-0004</td>
			<td>Stock concentration 10 mM 
			Final concentration 3 &#956;M</td>
		</tr>
		<tr>
			<td>Cholera toxin</td>
			<td>Sigma</td>
			<td>C8052</td>
			<td>Stock concentration 0.3 mg/mL 
			Final concentration 20 pM</td>
		</tr>
		<tr>
			<td>Cy3 Goat anti-Rabbit IgG (H+L)</td>
			<td>Jackson Immunoresearch</td>
			<td>111-165-144</td>
			<td>Dilution of 1:500 to use</td>
		</tr>
		<tr>
			<td>DMEM (High glucose)</td>
			<td>HyClone</td>
			<td>SH30243.01</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>DMSO&#160;</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td>Stored at RT</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td>Stored at -20 &#176;C 
			Heat-inactivated before use</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma</td>
			<td>G9391</td>
			<td>Stored at RT</td>
		</tr>
		<tr>
			<td>GFP-PURO plasmids (Mitf, Sox10, Pax3, Sox2, Sox9 and Snai2)</td>
			<td>Hanheng Biological Technology Co., Ltd.</td>
			<td>pHBLPm003198 pHBLPm001143 pHBLPm002968 pHBLPm002981 pHBLPm004348 pHBLPm000325</td>
			<td>Stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Abcam</td>
			<td>ab220365</td>
			<td>Stored at RT</td>
		</tr>
		<tr>
			<td>Human EDN3</td>
			<td>American-Peptide</td>
			<td>88-5-10A</td>
			<td>Stock concentration 100 &#956;M 
			Final concentration 0.1 &#956;M</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma</td>
			<td>H0888</td>
			<td>Stock concentration 100 &#181;g/mL 
			Final concentration 0.5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>L-DOPA</td>
			<td>Sigma</td>
			<td>D9628</td>
			<td>Stored at RT</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td>Transfection reagent, stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Masson-Fontana staining kit</td>
			<td>Solarbio</td>
			<td>G2032</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Neutral balsam</td>
			<td>Solarbio</td>
			<td>G8590</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td>Stored at RT</td>
		</tr>
		<tr>
			<td>PBS (-)</td>
			<td>Gibco</td>
			<td>C10010500BT</td>
			<td>Stored at RT</td>
		</tr>
		<tr>
			<td>Phorbol 12-myristate 13-acetate (TPA)</td>
			<td>Sigma</td>
			<td>P8139</td>
			<td>Stock concentration 1 mM 
			Final concentration 200 nM</td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Sigma</td>
			<td>H9268</td>
			<td>cationic polymeric transfection reagent; Stock concentration 8 &#956;g/&#181;L 
			Final concentration 4 ng/&#181;L</td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Gibco</td>
			<td>A11138-03</td>
			<td>Stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Recombinant human bFGF</td>
			<td>Invitrogen</td>
			<td>13256-029</td>
			<td>Stock concentration 4 &#956;g/mL 
			Final concentration 10 ng/mL</td>
		</tr>
		<tr>
			<td>Recombinant human insulin</td>
			<td>Sigma&#160;</td>
			<td>I3536</td>
			<td>Stock concentration 10 mg/mL 
			Final concentration 5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Recombinant human SCF</td>
			<td>R&#38;D</td>
			<td>255-SC-010</td>
			<td>Stock concentration 200 &#956;g/mL 
			Final concentration 100 ng/mL</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sigma</td>
			<td>1330-20-7</td>
			<td>Stored at RT</td>
		</tr>
	</tbody>
</table>

direct reprogramming of mouse fibroblasts into melanocytes

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, there is no effective long-term treatment for vitiligo. Therefore, it is imperative to develop a convenient and effective treatment for vitiligo. Regenerative medicine technology for direct reprogramming of skin cells into melanocytes seems to be a promising novel treatment of vitiligo. This involves the direct reprogramming of the patient's skin cells into functional melanocytes to help ameliorate the loss of melanocytes in patients with vitiligo. However, this method needs to be first tested on mice. Although direct reprogramming is widely used, there is no clear protocol for direct reprogramming into melanocytes. Moreover, the number of available transcription factors is overwhelming. 
Here, a concentrated lentivirus packaging system protocol is presented to produce transcription factors selected for reprogramming skin cells to melanocytes, including Sox10, Mitf, Pax3, Sox2, Sox9, and Snai2. Mouse embryonic fibroblasts (MEFs) were infected with the concentrated lentivirus for all these transcription factors for the direct reprogramming of the MEFs into induced melanocytes (iMels) in vitro. Furthermore, these transcription factors were screened, and the system was optimized for direct reprogramming to melanocytes. The expression of the characteristic markers of melanin in iMels at the gene or protein level was significantly increased. These results suggest that direct reprogramming of fibroblasts to melanocytes could be a successful new therapeutic strategy for vitiligo and confirm the mechanism of melanocyte development, which will provide the basis for further direct reprogramming of fibroblasts into melanocytes in vivo.

This article includes the protocols of a concentrated lentivirus packaging system to produce lentivirus of transcription factors for direct reprogramming of fibroblasts to melanocytes and protocols for screening for transcription factors and direct reprogramming of melanocytes from MEFs.
The success of concentrated lentivirus production was evaluated by observing the fluorescence intensity of GFP (Figure 1A) or by flow cytometry (Figure 1B</st...

Watch this Scientific Journal Video about Direct Reprogramming of Mouse Fibroblasts into Melanocytes at JoVE.com

Direct Reprogramming of Mouse Fibroblasts into Melanocytes

Here, we describe an optimized direct reprogramming system for melanocytes and a high-efficiency, concentrated virus packaging system that ensures smooth direct reprogramming.

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, ...

Direct reprogramming to generate melanocytes has been reported. However, various transcription factors are used in different studies. In this protocol, we validated and reduced transcription factor number still with higher reprogramming efficiency.We established a direct reprogramming system to generate melanocytes using only three transcription factors, Mitf, Sox10 and PAX3, and modified the culture system to make it more stable and robust. How to regenerate functional melanocytes is a significant question that may help address therapy limitations for depigmentation diseases like vitiligo. The right reprogramming provides a prospective Our technique has the characteristics of convenience, strong operability, and stability, which is easier to promote and repeat.To begin the production of the concentrated lentivirus, plate 1.5 times 10 to the 6 HEK-293T cells into a 60-millimeter dish, and culture the cells with normal medium at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide. After 24 hours of the incubation, when the HEK-293T cells have reached 80 to 90%confluency, replace the medium with 3.5 mL of DMEM, then incubate the cells at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide for two hours. After removing the cells from the incubator, replace the medium with DMEM containing 2%FBS, and add the freshly prepared transfection complex mixture to the cells in a drop-wise manner.Mix the liquid mixture gently. At the completion of eight hours, change the cell medium with 3.5 mL of normal medium. Collect the virus supernatant at 24 and 48 hours.Next, mix the virus supernatants collected at two different time points and centrifuge the mixture at 200 xg for five minutes at four degrees Celsius. Then pass the collected supernatant through 0.45 micron filters to collect the filtrate in a 50-mL sterile conical tube. Concentrate the filtered virus supernatant by centrifugation at 6, 000 xg at four degrees Celsius overnight.Once done, ensure that the virus pellet is visible at the bottom of the conical tube. Pour out the supernatant slowly. For dissolving the virus pellet in the normal medium with one by 100th*volume of the virus supernatant.Use a P1000 micropipette to pipette up and down gently until a homogenous 100x concentrated virus mixture is obtained. Divide the concentrated virus into the microcentrifuge tubes to store at 80 degrees Celsius. Plate 1 times 10 to the 5th HEK-293T cells into one well of a six-well plate.Culture these cells with the normal medium at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide. After 24 hours, add 0.1 to 0.2 microliters of the 100x fluorescent concentrated virus to each well. Followed by the addition of four nanograms per microliters of the cationic polymeric transfection reagent to each well.About eight to twelve hours after infection with the virus, replace the medium with the normal medium. At forty-eight hours post-infection, wash the dish with 1 mL of sterile PBS to remove the dead cells. Then trypsinize the cells using 250 microliters of 0.05%trypsin-EDTA solution per well of a six-well plate at room temperature.Centrifuge the plate at 200 xg for five minutes at four degrees Celsius. After removing the supernatant, re-suspend the cell pellet in one mL of PBS and add the cell suspension to a five mL polystyrene round-bottom tube. Then place the tube into the flow cytometer to analyze the sample.For direct reprogramming of fibroblasts, coat one well of a six-well cell culture plate with 1 mL of 0.1%gelatin solution at room temperature. After 15 to 30 minutes, when the well is completely covered with the gelatin, aspirate 0.1%gelatin solution. Next, plate 5 times 10 to the 4th mouse embryonic fibroblasts, or MEFs, into one well of a six-well plate coated with 0.1%gelatin, and culture the cells overnight with the normal medium at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide.After 24 hours, when the MEFs have reached 40 to 50%confluency, replace the medium with the normal medium. Next, melt the frozen, concentrated virus on ice. Calculate the volume of the virus to be added using equation and then add the concentrated virus for the six transcription factors to each well, according to the calculated volume.Add four micrograms per mL of the cationic polymeric transfection agent to the well. Consider it as day zero of the lentivirus infection. On day one, after 8 to 12 hours of infection, replace the medium containing the virus with the fresh normal medium, while adding 0.5 micrograms per mL puromycin to screen stable infected cell lines.On day two, 48 hours after infection, replace the supernatant medium gradually with the reprogramming medium. Change one-fourth of the total medium volume before adding three micromolar CHIR99021. From day three to day seven, depending on the condition of the cells, change the medium by gradually replacing it with a higher proportion of reprogramming medium to switch to the complete reprogramming medium within five days.Next, detach the cells with 500 microliters of 0.05%trypsin-EDTA digestive enzyme for three minutes at room temperature. When approximately sixty percent of the cells have floated up, stop the digestion by adding the normal medium, two times the volume of the digestive enzyme. Collect the cell suspension in a 15-mL sterile conical tube to centrifuge at 200 xg for five minutes at four degree Celsius.Remove the supernatant before re-suspending the cell pellet with the reprogramming medium. When done, plate the re-suspended cells in a 60-millimeter sterile dish. Culture the cells at 37 degrees Celsius in a humidified 5%carbon dioxide incubator.After forty-eight hours of lentivirus infection in HEK-293T cells, the success of concentrated lentivirus production was evaluated by observing the fluorescence intensity of GFP or by flow cytometry. The titer of the concentrated virus was found to be high at 10 to the 8th TU/mL. During the direct reprogramming of the fibroblasts, the cell morphology changed gradually to elongated cell synapse and enlarged cell nuclei.However, the cells progressively aged after day twenty. Removal of the transcription factors, Mitf, Pax3 or Sox10, resulted in the silencing of the expression of melanocytic genes, Tyr, Tyrp1, and Mlana, indicating the greatest impact on fibroblast conversion to melanocytes. Hence, the expression of melanocytic genes induced by the direct programming with three transcription factors was higher when compared with six transcription factors.To identify MEF-induced melanocytes, or iMels, the expression of melanocytic markers, TYRP1 and DCT was detected using immunofluorescent staining. Moreover, the melanin-specific staining methods such as DOPA and Masson-Fontana staining showed positive results. The 293T cells should be distributed evenly.Also, the addition of transfection mixture to cells must be done gently to prevent the cells from floating. The protocol is stable and practical and it can be used to reprogram other types of cells. It expects to provide a reference and a strategy for in vivo direct reprogramming in melanocytes regeneration in future medicine.

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Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease phenotypes. Recent advances have identified more efficient and safe methods for introduction of reprogramming factors. However, there are few tools to monitor and track the progression of reprogramming. Current methods for monitoring reprogramming rely on the qualitative inspection of morphology or staining with stem cell-specific dyes and antibodies. Tools to dissect the progression of iPSC generation can help better understand the process under different conditions from diverse cell sources. 
This study presents key approaches for kinetic measurement of reprogramming progression using flow cytometry as well as real-time monitoring via imaging. To measure the kinetics of reprogramming, flow analysis was performed at discrete time points using antibodies against positive and negative pluripotent stem cell markers. The combination of real-time visualization and flow analysis enables the quantitative study of reprogramming at different stages and provides a more accurate comparison of different systems and methods. Real-time, image-based analysis was used for the continuous monitoring of fibroblasts as they are reprogrammed in a feeder-free medium system. The kinetics of colony formation was measured based on confluence in the phase contrast or fluorescence channels after staining with live alkaline phosphatase dye or antibodies against SSEA4 or TRA-1-60. The results indicated that measurement of confluence provides semi-quantitative metrics to monitor the progression of reprogramming.

The protocol presented in this study describes methods for the real-time monitoring of reprogramming progression via the kinetic measurement of positive and negative pluripotent stem cell markers using flow cytometry analysis. The protocol also includes the imaging-based assessment of morphology, and marker or reporter expression during iPSC generation.

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease ...

Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining the ability to self-renew and to differentiate into all subtypes of the trophoblast lineage, equivalent to the in vivo stem cell population giving rise to the fetal portion of the placenta. Therefore, TSCs offer a unique model to study placental development and embryonic versus extra-embryonic cell fate decision in vitro. From the blastocyst stage onwards, a distinct epigenetic barrier consisting of DNA methylation and histone modifications tightly separates both lineages. Here, we describe a protocol to fully overcome this lineage barrier by transient over-expression of trophoblast key regulators Tfap2c, Gata3, Eomes and Ets2 in murine embryonic fibroblasts. The induced trophoblast stem cells are able to self-renew and are almost identical to blastocyst derived trophoblast stem cells in terms of morphology, marker gene expression and methylation pattern. Functional in vitro and in vivo assays confirm that these cells are able to differentiate along the trophoblast lineage generating polyploid trophoblast giant cells and chimerizing the placenta when injected into blastocysts. The induction of trophoblast stem cells from somatic tissue opens new avenues to study genetic and epigenetic characteristics of this extra-embryonic lineage and offers the possibility to generate trophoblast stem cell lines without destroying the respective embryo.

Here we present a protocol for the direct conversion of murine embryonic fibroblasts into fully functional and stable trophoblast stem cells by ten day over-expression of Tfap2c, Gata3, Eomes and Ets2.

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining ...

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.

Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to ...

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). 
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic fluid and membrane mesenchymal stem cells, represent a unique type of undifferentiated cells with promise in tissue engineering and for reprogramming into iPSC for future pediatric interventions and stem cell banking. The protocol presented here describes an optimized procedure for extracting and culturing primary amniotic fluid and membrane mesenchymal stem cells and generating episomal induced pluripotent stem cells from these cells in fully chemically defined culture conditions utilizing human recombinant vitronectin and the E8 medium. Characterization of the new lines by applying stringent methods &#8211; flow cytometry, confocal imaging, teratoma formation and transcriptional profiling &#8211; is also described. The newly generated lines express markers of embryonic stem cells &#8211; Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 &#8211; while being negative for the SSEA-1 marker. The stem cell lines form teratomas in scid-beige mice in 6-8 weeks and the teratomas contain tissues representative of all three germ layers. Transcriptional profiling of the lines by submitting global expression microarray data to a bioinformatic pluripotency assessment algorithm deemed all lines pluripotent and therefore, this approach is an attractive alternative to animal testing. The new iPSC lines can readily be used in downstream experiments involving the optimization of differentiation and tissue engineering.

This protocol describes the reprogramming of primary amniotic fluid and membrane mesenchymal stem cells into induced pluripotent stem cells using a non-integrating episomal approach in fully chemically defined conditions. Procedures of extraction, culture, reprogramming, and characterization of the resulting induced pluripotent stem cells by stringent methods are detailed.

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.