The authors wish to thank Harene Ranjithakumaran and Stacey Jackson for technical support. This work was supported in part by grants from the National Health and Medical Research Council (R.C.B. Wong, A. P&#233;bay), the University of Melbourne (R.C.B. Wong), Retina Australia (R.C.B. Wong, S.S.C. Hung, A. P&#233;bay) and the Ophthalmic Research Institute of Australia (R.C.B. Wong, S.S.C. Hung, A. P&#233;bay); Australian Research Council Future Fellowship (A. P&#233;bay, FT140100047), Cranbourne Foundation Fellowship (R.C.B. Wong); intramural funding from the National Institutes for Health (R.C.B. Wong, S.S.C. Hung) and operational infrastructure support from the Victorian Government.

The collection of human hair sample from individuals requires ethical approval by the human research ethics committee in the host institutions and should be done in compliance with the institutional guidelines.
1. Isolation of Keratinocytes from Plucked Hair
<ol>
	<li>Thaw Extracellular Matrix (ECM) solution (i.e., Matrigel) on ice O/N.</li>
	<li>Using pre-chilled pipette tips, add 200 &#181;l&#160;ECM solution to 12 ml&#160;of chilled DMEM/F12 medium. Coat a 12-well plate with 1 ml&#160;of diluted ECM solution into each well. Incubate the coated plate O/N at 37 &#176;C. Wash the coated plate with pre-warmed DMEM/F12 medium before use.</li>
	<li>Collect primary human hair from the individual&#8217;s head by placing fingers close to the root of the hair and plucking the hair in one quick and smooth motion. Check to confirm that each collected hair contains an intact hair follicle and determine the growth phase of the hair under a dissecting microscope (Figure 1).
	<ol>
		<li>Collect 5-10 anagen hairs from each individual to extract keratinocytes. Process plucked hairs as soon as possible to ensure successful derivation of keratinocytes. 
		Note: Figure 1 illustrates the morphology of hairs in different phases of the growth cycle. Anagen hair is in the growth phase, with a visible outer layer of epithelium surrounding the length of the hair, known as the ORS. On the other hand, telogen hair represents hair in the resting phase and does not have a visible ORS, but has a clear ball of cells covering the root of the hair, known as the telogen bulge.</li>
	</ol>
	</li>
	<li>Place the hair samples into a petri dish containing 10 ml&#160;of Antibiotic Mix to wash for 5 min&#160;at RT.</li>
	<li>Using sterilized scissors and forceps, cut off excessive hair shaft, leaving a hair fragment with an intact hair follicle and around 0.5 - 1 cm of hair shaft.</li>
	<li>Carefully transfer 1 hair fragment into each well of the ECM-coated plate using forceps.</li>
	<li>Using a 1 ml&#160;pipette, add approximately 3 drops of Knockout Serum Replacement (KSR) medium (~100 - 200 &#181;l) to the hair samples. 
	Note: Keep the hair fragments in close contact to the plate surface to allow for attachment and subsequent keratinocyte outgrowth. Adding excessive media at this step may causes the hair fragments to float and decreases the chance of successful hair attachment.</li>
	<li>Incubate the hair samples in a 37 &#176;C incubator with 5% CO2 and allow the hair follicles to attach O/N. On the following day, gently add another 3 drops of KSR medium (~100 - 200 &#181;l) to keep the hair samples moist.</li>
	<li>Observe the hair samples daily to confirm that the hair follicles have attached successfully. Generally attachment of hair follicles is apparent within 1-3 days. Thick hairs are usually easier to attach compared to fine hairs, as fine hairs have higher tendency to float which hinder the attachment process.
	<ol>
		<li>Once the hair have attached to the coated plate, add 500 &#181;l&#160;of KSR medium to the well. Take extra care when adding the medium as hair samples may not have attached firmly. From this point, change media every 2 days. 
		Note: Keratinocyte outgrowths should be visible from the ORS region of the hair fragment after 3-7 days (Figure 2A-B). Allow the keratinocytes to grow up to 14 days before passaging the cells as described in the next section.</li>
	</ol>
	</li>
</ol>
2. Maintenance and Passaging of Keratinocytes
<ol>
	<li>Pre-coat a 6-well plate using the Coating Matrix Kit. Add 680 &#181;l&#160;of the dilution medium from the kit to each well, followed by 6.8 &#181;l&#160;of Coating Matrix solution. Shake the plate to ensure uniform distribution of the Coating Matrix solution on the plate.</li>
	<li>Allow the coated plate to incubate for 30 min&#160;at RT. Remove the Coating Matrix solution prior to plating of keratinocytes.</li>
	<li>Dissociate keratinocytes into a single cell suspension with 500 &#181;l&#160;of 0.25% trypsin per well for 2-5 min&#160;at 37 &#176;C. Inactivate the trypsin with 500 &#181;l&#160;of medium containing serum (e.g., Fetal Bovine Serum (FBS) medium) and collect into a sterile 15 ml&#160;tube. Remove the hair fragment using sterilized forceps.</li>
	<li>Determine the number of harvested cells using a haemocytometer.</li>
	<li>Centrifuge the cells for 3 min&#160;at 200 x g. Wash the cells with PBS and repeat centrifugation. Aspirate the PBS and resuspend the cell pellet in fresh Keratinocyte medium.</li>
	<li>Plate down 4 &#215; 104 keratinocytes into one well of ECM-coated plate in the presence of Keratinocyte medium. Keep the keratinocytes in a 37 &#176;C incubator with 5% CO2 (Figure 2C). Change media every day and passage cells when the culture is ~80% confluent, which may take approximately 2-7 days depending on the passage number and proliferation rate of the keratinocytes. The derived keratinocytes should be expanded to generate sufficient frozen stocks using standard slow-freezing cryopreservation methods16. 
	Note: The described culture conditions support the expansion of homogenous population of keratinocytes with typical cobblestone morphology (Figure 2C). Occasionally, a few differentiated keratinocytes, with large and flat morphology and have lost replicative potential, may be observed in culture. Characterization of keratinocytes can be performed by immunostaining using antibodies against cytokeratin 14 as previously described 17. Established keratinocyte cell lines should also be tested for mycoplasma after 1-3 passages using commercially available kit.</li>
</ol>
3. Generation of hiPSCs from Keratinocytes Using Episomal Vectors
<ol>
	<li>Pre-coat a 6-well plate with 0.1% gelatin (2 ml/well). Allow gelatin to coat for at least 20 min&#160;at RT.</li>
	<li>For reprogramming experiment, use keratinocytes no later than passage 6. Dissociate keratinocytes into a single cell suspension with 1 ml&#160;of 0.25% trypsin per well for 2-5 min&#160;at 37 &#176;C. Inactivate trypsin with 1 ml&#160;of media containing serum (e.g., FBS media) and collect in a 15 ml&#160;tube.</li>
	<li>Centrifuge cells for 3 min&#160;at 200 x g and resuspend cells in Keratinocyte medium. Plate 1 &#215; 105-1.5 &#215; 105 keratinocytes per well in a gelatinized plate O/N.</li>
	<li>On the following day (Day 0), check the plate to ensure cells are 50-60% confluent. 
	Note: If keratinocytes are less than ~50% confluent they might not grow well after transfection.</li>
	<li>On Day 0, perform transfection of the episomal reprogramming vectors on the keratinocytes, using a ratio of 8 &#181;l&#160;transfection reagent to 2 &#181;g total plasmid DNA.
	<ol>
		<li>Take 0.5 &#181;g for each of the 4 plasmids (pCXLE-eGFP, pCXLE-hOct3/4-shP53F, pCXLE-hSK, pCXLE-hUL) and dilute in 100 &#181;l&#160;of Reduced Serum medium (e.g., OPTI-MEM). 
		Note: The plasmid pCXLE-eGFP is used to monitor the transfection efficiencies in keratinocytes and is not required for successful reprogramming.</li>
		<li>Add 8 &#181;l&#160;of transfection reagent to the diluted DNA. Mix by flicking the tube. Allow the DNA reaction mix to rest undisturbed at RT for 15 min.</li>
		<li>Replace the Keratinocyte medium with fresh medium.</li>
		<li>Gently add the DNA reaction mix to keratinocyte culture and incubate in a 37 &#176;C incubator with 5% CO2 for 4 hr. 
		Note: It is not recommended to perform prolonged transfection O/N in keratinocytes as they exhibit low survival post-transfection.</li>
		<li>After 4 hr&#160;post-transfection, replace the Keratinocyte medium with fresh medium.</li>
	</ol>
	</li>
	<li>On Day 1 (1 day post-transfection), change the keratinocyte medium and check transfection efficiency by estimating the percentage of eGFP positive cells under a fluorescent microscope. Typically &#62; 60-70% transfection efficiency is observed for primary keratinocytes using this protocol.</li>
	<li>On Day 2, repeat transfection as described in step 3.5. Two consecutive transfections would typically achieve &#62; 90% transfection efficiency. Keratinocyte culture usually reaches confluence by the end of the second transfection.
	<ol>
		<li>Prepare a 10 cm dish of mouse embryonic fibroblast (MEF) feeder for each reprogramming reaction. Prepare mitotically inactivated MEF feeders as previously described 18.</li>
		<li>Pre-coat a 10 cm dish with 5 ml&#160;of 0.1% gelatin for at least 20 min. Plate 4 &#215; 106 mitotically inactivated MEF per 10 cm dish in FBS medium. Allow the cells to settle and attach O/N in a 37 &#176;C incubator with 5% CO2.</li>
	</ol>
	</li>
	<li>On Day 3, harvest the keratinocytes with 0.25% trypsin as described in step 3.2 and 3.3. Resuspend the keratinocytes in KSR medium and plate 90% of keratinocytes from 1 well of a 6-well plate into a 10 cm MEF feeder dish with KSR medium.</li>
	<li>From Day 4 onwards, change the KSR medium every day.
	<ol>
		<li>Alternatively after Day 18, replace culture medium every second day. 
		Note: Non-reprogrammed keratinocytes will cease to proliferate, while hiPSC colonies with defined boundary that resemble human embryonic stem cells (hESCs) will emerge from Day 14 - 21.</li>
	</ol>
	</li>
	<li>Isolate hiPSC colonies by manual dissection from Day 21 onwards. Avoid hiPSC colonies that are closely clustered together and only pick hiPSC colonies that are clearly separated from others. 
	Note: hiPSC colonies can be picked earlier, &#160;but the colony with defined boundaries might not be obvious at earlier time points given the small colony size.&#160;Reprogramming efficiencies may vary between different patient&#8217;s keratinocytes, and is also influenced by other factors such as passage number or proliferation rate of keratinocytes.
	<ol>
		<li>For manual dissection of hiPSC colonies, use a 26G needle to cut hiPSC colonies under a dissecting microscope. Cut a hiPSC colony into small pieces around 300 - 600 &#181;m in length. Transfer the pieces from one hiPSC colony into a new MEF feeder plate (2.5 &#215; 104 MEF/cm2) to establish a clonal line of hiPSCs. Initially, culture each hiPSC clonal line in one well of a 12 well plate or organ culture dishes, then expand to 6 well plate format.</li>
		<li>Maintain established clonal lines of hiPSCs in KSR medium on MEF feeders. Once the culture reach 70 - 80% confluency with hiPSC colonies ~1.5 mm in diameter, passage hiPSCs using standard enzymatic passaging methods (Accutase, Dispase or Collagenase IV) according to the manufacturer&#8217;s instruction.</li>
	</ol>
	</li>
	<li>Characterize established hiPSC lines to confirm pluripotency and their ability to differentiate into cells representative of the three germ layers in vitro and/or in vivo13,19. Also, routinely test established hiPSC lines for mycoplasma using commercially available kits to ensure they are pathogen-free. Routinely monitor genomic stability of hiPSC lines &#160;by karyotyping or array-based copy number variation (CNV) analysis.</li>
</ol>

<ol>
	<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=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Takahashi, K., 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=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Yu, J., 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=Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells.">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318, 1917-1920 (2007).</li><li>Okita, K., 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=A+more+efficient+method+to+generate+integration-free+human+iPS+cells.">A more efficient method to generate integration-free human iPS cells.</a> Nat Methods. 8, 409-412 (2011).</li><li>Park, I. 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=Reprogramming+of+human+somatic+cells+to+pluripotency+with+defined+factors.">Reprogramming of human somatic cells to pluripotency with defined factors.</a> Nature. 451, U141-U141 (2008).</li><li>Okita, K., 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=An+efficient+nonviral+method+to+generate+integration-free+human-induced+pluripotent+stem+cells+from+cord+blood+and+peripheral+blood+cells.">An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells.</a> Stem Cells. 31, 458-466 (2013).</li><li>Dowey, S. N., Huang, X., Chou, B. K., Ye, Z., Cheng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+integration-free+human+induced+pluripotent+stem+cells+from+postnatal+blood+mononuclear+cells+by+plasmid+vector+expression.">Generation of integration-free human induced pluripotent stem cells from postnatal blood mononuclear cells by plasmid vector expression.</a> Nat Protoc. 7, 2013-2021 (2012).</li><li>Zhou, T., 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=Generation+of+induced+pluripotent+stem+cells+from+urine.">Generation of induced pluripotent stem cells from urine.</a> J Am Soc Nephrol. 22, 1221-1228 (2011).</li><li>Aasen, T., 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=Efficient+and+rapid+generation+of+induced+pluripotent+stem+cells+from+human+keratinocytes.">Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes.</a> Nat Biotechnol. 26, 1276-1284 (2008).</li><li>Peters, A., Zambidis, E., Ye, K., Jin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+16.+Generation+of+nonviral+integration-free+induced+pluripotent+stem+cells+from+plucked+human+hair+follicles.">Chapter 16. Generation of nonviral integration-free induced pluripotent stem cells from plucked human hair follicles.</a> Human Embryonic and Induced Pluripotent Stem Cells: Lineage-Specific Differentiation Protocols.Springer Protocols Handbooks. , 203-227 (2012).</li><li>Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scratching+the+surface+of+skin+development.">Scratching the surface of skin development.</a> Nature. 445, 834-842 (2007).</li><li>Limat, A., Noser, F. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+cultivation+of+single+keratinocytes+from+the+outer+root+sheath+of+human+scalp+hair+follicles.">Serial cultivation of single keratinocytes from the outer root sheath of human scalp hair follicles.</a> J Invest Dermatol. 87, 485-488 (1986).</li><li>Piao, Y., Hung, S. S., Lim, S. Y., Wong, R. C., Ko, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+integration-free+human+induced+pluripotent+stem+cells+from+keratinocytes+by+simple+transfection+of+episomal+vectors.">Efficient generation of integration-free human induced pluripotent stem cells from keratinocytes by simple transfection of episomal vectors.</a> Stem Cells Transl Med. 3, 787-791 (2014).</li><li>Higgins, C. A., 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=Reprogramming+of+human+hair+follicle+dermal+papilla+cells+into+induced+pluripotent+stem+cells.">Reprogramming of human hair follicle dermal papilla cells into induced pluripotent stem cells.</a> J Invest Dermatol. 132, 1725-1727 (2012).</li><li>Muchkaeva, I. A., 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=Generation+of+iPS+Cells+from+Human+Hair+Follice+Dermal+Papilla+Cells.">Generation of iPS Cells from Human Hair Follice Dermal Papilla Cells.</a> Acta naturae. 6, 45-53 (2014).</li><li>Naaldijk, Y., Friedrich-Stockigt, A., Sethe, S., Stolzing, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+different+cooling+rates+for+fibroblast+and+keratinocyte+cryopreservation.">Comparison of different cooling rates for fibroblast and keratinocyte cryopreservation.</a> J Tissue Eng Regen. , (2013).</li><li>Sporl, F., 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=Real-time+monitoring+of+membrane+cholesterol+reveals+new+insights+into+epidermal+differentiation.">Real-time monitoring of membrane cholesterol reveals new insights into epidermal differentiation.</a> J Invest Dermatol. 130, 1268-1278 (2010).</li><li>Conner, D. A., et al., Ausubel, F. N., 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=Mouse+embryo+fibroblast+(MEF)+feeder+cell+preparation.">Mouse embryo fibroblast (MEF) feeder cell preparation.</a> Current protocols in molecular biology. 23, Unit 23 22 (2001).</li><li>Pebay, A., 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=Essential+roles+of+sphingosine-1-phosphate+and+platelet-derived+growth+factor+in+the+maintenance+of+human+embryonic+stem+cells.">Essential roles of sphingosine-1-phosphate and platelet-derived growth factor in the maintenance of human embryonic stem cells.</a> Stem Cells. 23, 1541-1548 (2005).</li><li>Myung, P., Ito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+bulge+in+hair+regeneration.">Dissecting the bulge in hair regeneration.</a> J Clin Invest. 122, 448-454 (2012).</li><li>Alonso, L., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hair+cycle.">The hair cycle.</a> J Cell Sci. 119, 391-393 (2006).</li><li>Robinton, D. A., Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promise+of+induced+pluripotent+stem+cells+in+research+and+therapy.">The promise of induced pluripotent stem cells in research and therapy.</a> Nature. 481, 295-305 (2012).</li><li>Soares, F. A., Sheldon, M., Rao, M., Mummery, C., Vallier, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+coordination+of+large-scale+human+induced+pluripotent+stem+cell+initiatives:+Wellcome+Trust+and+ISSCR+workshops+white+paper.">International coordination of large-scale human induced pluripotent stem cell initiatives: Wellcome Trust and ISSCR workshops white paper.</a> Stem cell reports. 3, 931-939 (2014).</li><li>McKernan, R., Watt, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+the+point+of+large-scale+collections+of+human+induced+pluripotent+stem+cells.">What is the point of large-scale collections of human induced pluripotent stem cells.</a> Nat Biotechnol. 31, 875-877 (2013).</li><li>Utikal, J., 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=Immortalization+eliminates+a+roadblock+during+cellular+reprogramming+into+iPS+cells.">Immortalization eliminates a roadblock during cellular reprogramming into iPS cells.</a> Nature. 460, U1145-U1112 (2009).</li><li>Xu, Y., 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=Proliferation+rate+of+somatic+cells+affects+reprogramming+efficiency.">Proliferation rate of somatic cells affects reprogramming efficiency.</a> J Biol Chem. 288, 9767-9778 (2013).</li><li>Liu, J., 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=Late+passage+human+fibroblasts+induced+to+pluripotency+are+capable+of+directed+neuronal+differentiation.">Late passage human fibroblasts induced to pluripotency are capable of directed neuronal differentiation.</a> Cell Transplant. 20, 193-203 (2011).</li><li>Huallachain, M., Karczewski, K. J., Weissman, S. M., Urban, A. E., Snyder, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+genetic+variation+in+somatic+human+tissues.">Extensive genetic variation in somatic human tissues.</a> Proc Natl Acad Sci U S A. 109, 18018-18023 (2012).</li><li>Abyzov, A., 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=Somatic+copy+number+mosaicism+in+human+skin+revealed+by+induced+pluripotent+stem+cells.">Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells.</a> Nature. 492, 438-442 (2012).</li></ol>

The authors have no conflict of interest.

Generation of patient-specific hiPSCs offers a unique approach for studying pathogenesis in the diseased cell types in vitro, and also provides a platform for drug screening to identify novel molecules that can rescue the disease phenotypes. This disease modeling approach using hiPSCs has yielded promising results for a variety of diseases, including Long QT syndrome, Huntington&#8217;s disease, Parkinson&#8217;s disease and amyotrophic lateral sclerosis 22. Several initiatives are already underway to...

The discovery of human induced pluripotent stem cells (hiPSCs) has revolutionized the field of regenerative medicine, providing a feasible method for generation of patient-specific stem cells 1-3. hiPSCs have been successfully generated from various somatic cell types, including fibroblasts 4,5, hematopoietic cells 6,7, renal epithelial cells from urine 8 and keratinocytes 9,10. To date, skin fibroblasts and hematopoietic cells represent the most commonly used cell sources for generating patient-specific iPSCs. Arguably, this is due to the fact that skin biopsies and blood collection are routine medical procedures and large biobanks of patient blood or skin samples have been established in many countries.
In contrast to blood cells and skin fibroblast which require invasive extraction methods, keratinocytes represent an easily accessible cell type for hiPSC generation. Keratinocytes are keratin-rich epithelial cells that form the exterior epidermal barrier of the skin and are also found in nails and hair 11. In particular, keratinocytes can be found on the outer root sheath (ORS) of hair follicles, an external cellular layer that covered the hair shaft together with the inner root sheath (IRS) cells (12, Figure 1). As hair collection is a simple procedure that does not require the assistance of medical personnel, it provides an opportunity for patients to collect and send their own hair samples to laboratories, which would greatly facilitate the collection of patient samples for hiPSC generation. Epidermal keratinocytes also have a higher reprogramming efficiency and faster reprogramming kinetics compared to fibroblasts, adding to the advantages of using keratinocytes as the starting cells for hiPSC generation 9,13. Furthermore, hiPSCs can also be generated using other cell populations within the hair follicle, including the dermal papilla cells located at the base of the hair follicle 14,15.
Previous reports of iPSC generation using hair-derived cells often utilize retroviral or lentiviral-based reprogramming methods 9,14,15. However, these viral methods introduce undesirable genomic integration of foreign transgenes during reprogramming. In comparison, the use of episomal vectors represents a feasible, non-viral reprogramming method to generate integration-free iPSCs 4. We have previously developed a simple, cost-effective and non-viral method to efficiently reprogram keratinocyte into hiPSCs using episomal vectors 13. Here we provide a detailed protocol for the generation of keratinocyte-derived hiPSCs, including the derivation of keratinocytes from plucked hair, expansion and maintenance of the keratinocytes and subsequent reprogramming to generate hiPSCs.

<table><tbody><tr><td>Antibiotic Mix:&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>250 ng/ml Antimycotic amphotericin B</td><td>Sigma</td><td>A2942-20ml</td><td>Antibiotic mix is made up in PBS.&amp;nbsp;</td></tr><tr><td>1X Penicillin/Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>PBS (-)</td><td>Invitrogen</td><td>14190-144</td><td></td></tr><tr><td>Knockout Serum Replacement (KSR) medium:&amp;nbsp;</td><td></td><td></td><td>KSR medium is filtered using Stericup (Millipore, #SCGPU05RE) before use. bFGF is added fresh to the media before use.</td></tr><tr><td>20% knockout serum replacement (KSR)</td><td>Invitrogen</td><td>10828-028</td><td></td></tr><tr><td>DMEM/F12 with glutamax</td><td>Invitrogen</td><td>10565-042</td><td></td></tr><tr><td>1&amp;times; MEM non-essential amino acid</td><td>Invitrogen</td><td>11140-050</td><td></td></tr><tr><td>&amp;nbsp;0.5&amp;times; Penicillin/Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>&amp;nbsp;0.1 mM &amp;beta;-mercaptoethanol</td><td>Invitrogen</td><td>21985</td><td></td></tr><tr><td>&amp;nbsp;bFGF (10 ng/ml, added fresh)</td><td>Millipore</td><td>GF003</td><td></td></tr><tr><td>Keratinocyte medium:&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;EpiLife with 60&amp;nbsp;&amp;micro;M Calcium</td><td>Invitrogen</td><td>M-EPI-500-CA</td><td></td></tr><tr><td>1&amp;times; Human keratinocyte growth supplement (HKGS)</td><td>Invitrogen</td><td>S-001-5</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS) medium:&amp;nbsp;</td><td></td><td></td><td>FBS medium is filtered using Stericup (Millipore, #SCGPU05RE) before use.</td></tr><tr><td>10% fetal bovine serum (FBS)</td><td>Invitrogen</td><td>26140079</td><td></td></tr><tr><td>DMEM&amp;nbsp;</td><td>Invitrogen</td><td>11995-073</td><td></td></tr><tr><td>0.5x Penicillin/Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>2 mM L-glutamine</td><td>Invitrogen</td><td>25030</td><td></td></tr><tr><td>0.25% trypsin-EDTA</td><td>Invitrogen</td><td>25200-056</td><td></td></tr><tr><td>Extracellular Matrix (ECM):</td><td></td><td></td><td></td></tr><tr><td>Matrigel</td><td>Corning&amp;nbsp;</td><td>354234</td><td>Aliquot Matrigel stock and store in -80&amp;deg;C following manufacturer&amp;rsquo;s instructions. Stock concentration of Matrigel varies slightly from batch to batch (~9mg/ml). We recommend to use 200&amp;micro;l matrigel for coating a 12-well plate (~150&amp;micro;g/well).&amp;nbsp;</td></tr><tr><td>Coating Matrix Kit&amp;nbsp;</td><td>Invitrogen</td><td>R-011-K</td><td></td></tr><tr><td>Plasmids:&amp;nbsp;</td><td></td><td></td><td>Note that pCXLE-eGFP is only used for monitoring transfection efficiency and is not required for reprogramming.</td></tr><tr><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; pCXLE-eGFP</td><td>Addgene</td><td>27082</td><td></td></tr><tr><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; pCXLE-hOct3/4-shP53F</td><td>Addgene</td><td>27077</td><td></td></tr><tr><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; pCXLE-hSK</td><td>Addgene</td><td>27078</td><td></td></tr><tr><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; pCXLE-hUL</td><td>Addgene</td><td>27080</td><td></td></tr><tr><td>Transfection reagent Fugene HD</td><td>Promega</td><td>E231B</td><td></td></tr><tr><td>Gelatin (from porcine skin)</td><td>Sigma</td><td>G1890</td><td>Make up 0.1% gelatin in distilled water. Autoclave before use.&amp;nbsp;</td></tr><tr><td>Reduced Serum medium: OPTI-MEM</td><td>Invitrogen</td><td>31985062</td><td></td></tr><tr><td>Accutase</td><td>Sigma</td><td>A6964-100ml</td><td></td></tr><tr><td>Mouse embryonic fibroblast (MEF) feeder</td><td></td><td></td><td>MEF can be inactivated by mitomycin C treatment or irradiation as described previously 16.</td></tr><tr><td>26G needle</td><td>Terumo</td><td>NN2613R</td><td></td></tr><tr><td>6-well plate (tissue culture treated)</td><td>BD Biosciences</td><td>353046</td><td></td></tr><tr><td>12-well plate (tissue culture treated)</td><td>BD Biosciences</td><td>353043</td><td></td></tr><tr><td>10 cm dish (tissue culture treated)</td><td>BD Biosciences</td><td>353003</td><td></td></tr><tr><td>Dispase</td><td>Invitrogen</td><td>17105-041</td><td>Use at 10 mg/ml</td></tr><tr><td>Collagenase IV</td><td>Invitrogen</td><td>17104-019</td><td>Use at 1 mg/ml</td></tr><tr><td>TRA-160 antibody</td><td>Millipore</td><td>MAB4360</td><td>Use at 5 &amp;micro;g/ml</td></tr><tr><td>OCT4 antibody</td><td>Santa Cruz</td><td>SC-5279</td><td>Use at 5 &amp;micro;g/ml</td></tr><tr><td>NANOG antibody</td><td>R&amp;amp;D Systems</td><td>AF1997</td><td>Use at 10 &amp;micro;g/ml</td></tr><tr><td>MycoAlert Detection kit</td><td>Lonza</td><td>LT07-418</td><td></td></tr></tbody></table>

generation of integration-free human induced pluripotent stem cells using hair-derived keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

The hair goes through 3 different phases of growth cycle: anagen (the growth phase), catagen (the regression phase) and telogen (the rest phase) 20,21. The anagen hair follicle contains multiple layers of epithelium; these layers include the ORS, IRS and the hair shaft (Figure 1). Anagen hair eventually undergoes transition to the catagen phase, which is marked by apoptosis of the ORS and termination of hair shaft differentiation. Finally, catagen hair transition to the telogen phase, where ap...

Watch this Scientific Journal Video about Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes at JoVE.com

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

generation-integration-free-human-induced-pluripotent-stem-cells

Research

JoVE Journal

Developmental Biology

11.4K Views.  University of Melbourne. This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors. 

Video: Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Neuroscience

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS cell cultures to feeder-free while still maintaining your use of Knockout SR.

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

Biology

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS and hES cell cultures to feeder-free while still maintaining your use of Knockout SR.

This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...