Name: Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles (Video) | JoVE Journal
Uploaded: 2013-04-06T14:00:00.000+00:00
Duration: 7 min
Description: Learn to isolate and culture neural crest stem cells from human hair follicles using enzymatic digestion and flow cytometry. This protocol enables researchers to obtain CD271+ HNK1+ cells for regenerative medicine studies.

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5 minutes

This work is supported by NIH grant R01AR054593 and R01AR054593-S1 to Xu.

1. Preparation of Tissue Culture Plates
<ol>
 <li>Coat each well with enough Poly-D-Lysine (PDL) to cover the bottom of the well. Allow the plates to dry in the hood.</li>
 <li>After the wells are dry, rinse with sterile water, and aspirate. Allow the plates to dry in the hood.</li>
 <li>When dry, coat with fibronectin (that was dissolved in biowhittaker water overnight at 37 °C at a concentration of 1 mg in 6 ml).</li>
 <li>Add NCSC medium [95 ml DMEM/F12, 1 ml Penn/Strep (P/S), 1 ml N2, 2 ml B27, 100 μl mercaptoethanol (2ME; 50 mM stock), bFGF (20 ng/ml medium), IGF-1 (20 ng/ml of medium) and EGF(20 ng/ml of medium)] before fibronectin dry in the plates,</li>
</ol>
2. Extract Hair Follicle Cells from Human Scalp
<ol>
 <li>Before starting the procedure of isolating NCSCs, one needs to prepare the respective medias and reagents (see Table 1).</li>
 <li>Fresh adult human scalp skin from facelift procedures or fetal scalp tissue is collected, washed with PBS containing Penicillin-Streptomycin.</li>
 <li>The skin is then transferred into 50 ml tubes and incubated in DMEM with Dispase (10 mg/ml) overnight at 4 °C. Incubation for 2-4 hr at 37 °C is also effective. Skin pieces should be a maximum width of 1 cm to allow for enzyme to penetrate.</li>
 <li>Transfer the skin into a sterilized Petri dish; pull off each hair from the skin by grasping the hair shaft near the skin surface and pulling firmly and smoothly. The hair follicles show morphology of either anagen (Figure 1A) or telogen (Figure 1B).</li>
 <li>Incubate the isolated follicle fragments in 0.05% trypsin-EDTA (Invitrogen) for 15-20 min at room temperature with shaking periodically, add 4 ml DMEM with 10% FBS to stop reaction. The follicular epithelium is trypsinized and filtered through 40 μm filter to obtain a single-cell suspension containing cells of varying size and shape.</li>
 <li>Spin at 200 x g for 5 min, discard supernatant carefully, re-suspend in 1 ml PBS containing 2% serum (FBS).</li>
 <li>Alternatively, plucked hair follicles can be placed in culture without trypsin digestion to grow hair spheres in situ.</li>
</ol>
3. Isolation of Hair Follicle NCSCs Using Flow Cytometric Cell Sorting
<ol>
 <li>Hair follicle single-cell suspension were obtained by trypsin digestion and labeled with antibodies against CD271 (APC-conjugated) /HNK1 (FITC-conjugated) or CD271/ alpha4 integrin (PE-conjugated) for 40 min on ice in the dark.</li>
 <li>Centrifuge for 5 min at 200 x g at room temperature and aspirate the supernatant.</li>
 <li>Resuspend cells in PBS containing 2% serum (FBS), and before sorting, PI is added to gate out the dead cells.</li>
 <li>Perform cell sorting by flow cytometry (FACS). Collect CD271+, HNK1+ double positive cells or CD271+, alpha4 integrin+ double positive cells (Figure 2).</li>
 <li>After sorting, CD271+, HNK1+ double positive cells or CD271+, alpha4 integrin+ double positive cells are cultured in ultra-low attachment plates in the NCSC medium</li>
</ol>
4. Culture of Primary NCSCs
<ol>
 <li>Culture the disassociated follicular cells or FACS sorted cells in ultra-low attachment plates in the NCSC medium, change medium every day.</li>
 <li>Check cell culture every day under microscope. NCSCs will start to form floating small aggregates after several days and well-formed spheres in 2-5 weeks in the culture depending on age of the donors (Figure 3A). Outgrowth will appear in a few days at the bulge region and well-formed spheres in situ in several weeks (Figure 3B) when entire hair follicles are cultured in the NCSC medium.</li>
</ol>
5. Expansion of NCSCs
<ol>
 <li>Wash cells with PBS once.</li>
 <li>Add pre-warmed (to 37 °C, critical) accutase and incubate at 37 °C for 5-10 min with shaking periodically to make sure Neural stem cell spheres dissociated into single cell( for attachment cultured NCSC, incubate cells with accutase at 37 °C for 5 min to make sure hair follicle stem cells detached).</li>
 <li>Wash and centrifuge cells twice for 5 min at 200 x g at room temperature in DMEM/F12 medium to remove any remaining accutase solution.</li>
 <li>Re-suspend cells with NCSC culture medium.</li>
 <li>For floating culture, put the cells in ultra-low attachment plates. For attachment culture, put the cells in pretreated tissue culture plates.</li>
</ol>
6. Representative Results
NCSC culture medium is sufficient to maintain hNCSCs in an undifferentiated state without the need for feeder cells. Keratinocytes will not proliferate and gradually died in the medium. Certain small round cells will proliferate and form small aggregates in suspension after 3 to 5 days. These floating aggregates slowly increased in size, generated three-dimensional sphere-like structures, which we termed hair spheres (Figure 3A). If cultured in coated plates, the NCSCs will attach to the surface and not form any spheres. The attached NCSCs grow faster than in suspension. The sphere-forming or attached stem cells express NCSCs markers. When entire hair follicles are cultures, spheres are formed at the area corresponding to the bulge region (Figure 3B) .
<img alt="Microscope image of fiber structure analysis, showing crystallization and surface morphology comparison." src="/files/ftp_upload/3194/3194fig1.jpg" data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. Plucked anagen (A) and telogen (B) hair follicles.
 
<img alt="Flow cytometry results; CD271 vs. alpha 4 integrin and HNK1 scatter plots, cell analysis." src="/files/ftp_upload/3194/3194fig2.jpg" data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Representative images of FACS analysis of NCSCs. Left panel: cells are gated using anti-CD271 and anti-alphe 4 integrin antibodies. Right panel: cells are gated using anti-CD271 and anti-HNK1 antibodies.
<img alt="Cell morphology phases; microscopy images A and B; stem cell aggregation and differentiation." src="/files/ftp_upload/3194/3194fig3.jpg" data-altupdatedat="1788235205000" data-alt="Figure 3"> 
Figure 3. Morphology of hair spheres. (A) Morphology of floating hair spheres, left panel: low power, right panel: high power. (B) Morphology of hair sphere in situ at the bulge region, left panel: a hair sphere at telogen bulge, right panel: a hair sphere at the bulge region of an anagen hair follicle.

<ol><li>Cotsarelis, G., Cheng, S. Z., Dong, G., Sun, T. T., Lavker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Existence+of+slow-cycling+limbal+epithelial+basal+cells+that+can+be+preferentially+stimulated+to+proliferate%3A+implications+on+epithelial+stem+cells%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells.</a> Cell. 57, 201-209 (1989).</li>
<li>Cotsarelis, G., Sun, T. T., Lavker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Label-retaining+cells+reside+in+the+bulge+area+of+pilosebaceous+unit%3A+implications+for+follicular+stem+cells%2C+hair+cycle%2C+and+skin+carcinogenesis%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis.</a> Cell. 61, 1329-1337 (1990).</li>
<li>Tanimura, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hair+follicle+stem+cells+provide+a+functional+niche+for+melanocyte+stem+cells%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Hair follicle stem cells provide a functional niche for melanocyte stem cells.</a> Cell Stem Cell. 8, 177-187 (2011).</li>
<li>Yu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+a+novel+population+of+multipotent+adult+stem+cells+from+human+hair+follicles%5BTitle%5D)+AND+%22Am.+J.+Pathol%22%5BJournal%5D)">Isolation of a novel population of multipotent adult stem cells from human hair follicles.</a> Am. J. Pathol. 168, 1879-1888 (2006).</li>
<li>Yu, H., Kumar, S. M., Kossenkov, A. V., Showe, L., Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stem+cells+with+neural+crest+characteristics+derived+from+the+bulge+region+of+cultured+human+hair+follicles%5BTitle%5D)+AND+%22J.+Invest.+Dermatol%22%5BJournal%5D)">Stem cells with neural crest characteristics derived from the bulge region of cultured human hair follicles.</a> J. Invest. Dermatol. 130, 1227-1236 (2010).</li>
<li>Wong, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+crest-derived+cells+with+stem+cell+features+can+be+traced+back+to+multiple+lineages+in+the+adult+skin%5BTitle%5D)+AND+%22J.+Cell+Biol%22%5BJournal%5D)">Neural crest-derived cells with stem cell features can be traced back to multiple lineages in the adult skin.</a> J. Cell Biol. 175, 1005-1015 (2006).</li>
<li>Sieber-Blum, M., Grim, M., Hu, Y. F., Szeder, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pluripotent+neural+crest+stem+cells+in+the+adult+hair+follicle%5BTitle%5D)+AND+%22Dev.+Dyn%22%5BJournal%5D)">Pluripotent neural crest stem cells in the adult hair follicle.</a> Dev. Dyn. 231, 258-269 (2004).</li>
<li>Amoh, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Implanted+hair+follicle+stem+cells+form+Schwann+cells+that+support+repair+of+severed+peripheral+nerves%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Implanted hair follicle stem cells form Schwann cells that support repair of severed peripheral nerves.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 17734-17738 (2005).</li>
<li>Koliatsos, V. E., Xu, L., Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+stem+cell+grafts+as+therapies+for+motor+neuron+disease%5BTitle%5D)+AND+%22Expert+Opin.+Biol.+Ther%22%5BJournal%5D)">Human stem cell grafts as therapies for motor neuron disease.</a> Expert Opin. Biol. Ther. 8, 137-141 (2008).</li>
<li>McKenzie, I. A., Biernaskie, J., Toma, J. G., Midha, R., Miller, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Skin-derived+precursors+generate+myelinating+Schwann+cells+for+the+injured+and+dysmyelinated+nervous+system%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Skin-derived precursors generate myelinating Schwann cells for the injured and dysmyelinated nervous system.</a> J. Neurosci. 26, 6651-6660 (2006).</li>
<li>LeDouarin, N. M., Kalcheim, C. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aLeDouarin%2C+NM+%22The+Neural+Crest%22">The Neural Crest</a>. , Cambridge University Press. Cambridge. (1999).</li>
<li>Tumbar, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defining+the+epithelial+stem+cell+niche+in+skin%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Defining the epithelial stem cell niche in skin.</a> Science. 303, 359-363 (2004).</li>
<li>Roh, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multi-potentiality+of+a+new+immortalized+epithelial+stem+cell+line+derived+from+human+hair+follicles%5BTitle%5D)+AND+%22In+Vitro+Cell+Dev.+Biol.+Anim%22%5BJournal%5D)">Multi-potentiality of a new immortalized epithelial stem cell line derived from human hair follicles.</a> In Vitro Cell Dev. Biol. Anim. 44, 236-244 (2008).</li>
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We have nothing to disclose.

The cell isolation and culture methods described are reproducible and robust. We have generated NCSCs from dozens of individuals across a broad age range. Although it is best to process the tissue right after tissue harvest, we found that scalp tissues can be safely stored in media on ice for overnight transportation with minimal impact on cell viability.
It is important to treat the scalp tissue with antibiotics and use aseptic technique during hair follicle isolation to avoid potential micro...

<table><tbody><tr><td>DMEM</td><td>Invitrogen</td><td>11965-092</td><td></td></tr><tr><td>DMEM/F12</td><td>Invitrogen</td><td>11330-32</td><td></td></tr><tr><td>Heat-inactivated FBS</td><td>Hyclone</td><td>SH30071.03</td><td></td></tr><tr><td>B27 supplement</td><td>Invitrogen</td><td>17504044</td><td></td></tr><tr><td>N2 supplement</td><td>Invitrogen</td><td>17502048</td><td></td></tr><tr><td>bFGF</td><td>Invitrogen</td><td>PHG0026</td><td></td></tr><tr><td>EGF</td><td>R&amp;amp;D system</td><td>236-EG-01M</td><td></td></tr><tr><td>IGF-I</td><td>R&amp;amp;D system</td><td>291-G1-050</td><td></td></tr><tr><td>0.05% Trypsin/EDTA</td><td>Invitrogen</td><td>25300-054</td><td></td></tr><tr><td>Dispase</td><td>Invitrogen</td><td>17105041</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Invitrogen</td><td>15070063</td><td></td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;&lt;strong&gt;Table 1.&lt;/strong&gt;&lt;/p&gt;</td></tr></tbody></table>

isolation and culture of neural crest stem cells from human hair follicles

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 ...

The overall goal of this procedure is to isolate and culture primary neural crest stem cells, or N CSCs from human scalp tissues. This is accomplished by first cussing human scalp skin into one centimeter wide strips and incubating in media containing dis bays. The skin is then arranged in the fresh media and the hair follicles are plugged.The second step of the procedure is to treat the hair follicles with trypsin and single cell suspensions are obtained. The final step of the procedure is to sort CD 2 71 HNK one double positive ncsc using flow cytometry and to plate in culture plates. Ultimately phase contrast microscopy is used to study neuros sphere formation and growth, Where demonstration of this methods is critical as the extracting and isolating health vertical steps are difficult to learn because it is very hard to dissect the skin and health particles under microscope.To prepare for culturing stem cells coat the bottom of each well of a six Well plate with poly de lycine allow the plates to dry in the hood. Once the wells are dry, rinse with sterile water and aspirate, then allow the wells to dry again. Coat the plates with the 0.17 milligram per milliliter solution of fibronectin.Then before the fibronectin dries in the plate at NCSC culture medium, prepare and have ready the following media and reagents. 97%D-M-E-M-F 12 2%B 27 supplement 1%and two supplement B-F-G-F-E-G-F IGF one and diss. Collect fresh adult human scalp skin from facelift procedures or fetal scalp tissue and wash it with PBS containing penicillin streptomycin.Transfer the skin into 50 milliliter tubes and incubated in DMEM with 10 milligrams per milliliter DIS space at 37 degrees Celsius for two to four hours or overnight at four degrees Celsius. Next, transfer the skin into a sterilized Petri dish and remove the hairs from the skin by grasping the hair shaft near the skin surface and pulling firmly and smoothly. The hair follicles will either show an antigen orgen morphology, incubate the isolated follicle fragments in 0.05%trips in EDTA for 15 to 20 minutes at room temperature with occasional shaking.Then add four milliliters of DMEM with 10%FBS to stop the reaction. Next, pass the follicular epithelium through a 40 micron filter to obtain a single cell suspension containing cells of varying size and shape. Then spin the suspension at 200 times G for five minutes.Carefully discard the S supernatant and resuspend the pellet in one milliliter of PBS with 2%FBS To isolate N CSCs using facts first, label the cells with antibodies against a PC conjugated CD 2 71 FE C conjugated hnk one or CD 2 71 PE conjugated alpha four integrin by incubating on ice for 40 minutes in the dark centrifuge at 200 times G for five minutes at room temperature and aspirate the supernatant. Re suspend the cells in PBS containing 2%FBS and two micrograms per milliliter. Propidium iodide to gate out the dead cells.Perform cell sorting by flow cytometry and collect CD 2 71 H and K one double positive or CD 2 71 alpha four integrin double positive cells culture. The cells in ultra-low attachment plates in NCSC medium, changing the medium once per day. Check the cell culture every day under the microscope.NCSC will start to form floating small aggregates after several days and well-formed spheres in two to five weeks in the culture, depending on the age of the donors. Outgrowth will appear in a few days at the bulge region and well-formed spheres in situ in several weeks when entire hair follicles are cultured in the NCSC medium. To expand NCSC cultures, wash the cells once with PBS prewarm two times tripsin EDTA to 37 degrees Celsius.Then add it to the cells for seven to 15 minutes for the hail follicle stem cell to detach. To stop the tripsin ADD DMEM with 10%FBS gently pipet up and down to disperse the cells. Then spin down at 200 times G for five minutes.Re suspend the cells in NCSC medium and culture and fibronectin coated plates at 37 degrees Celsius. NCSC culture. Medium is sufficient to maintain human NCSC in an undifferentiated state.Without the need for feeder cells, keratinocytes will not proliferate and gradually die in the medium. Some small round cells will proliferate and form small aggregates in suspension. After three to five days, these floating aggregates slowly increase in size generating three dimensional sphere like structures, termed hair spheres.When cultured encoded plates, the N CSCs attach to the surface and grow faster than sphere forming cells in suspension, both sphere forming and attached stem cells. Express NCSC markers when entire hair follicles are cultured spheres form at the area corresponding to the bulge region. After watching the video, you should have a good understanding of how to isolate and culture.Primary neuro stem cell from herma scalp tissues.

isolation-culture-neural-crest-stem-cells-from-human-hair

Research

JoVE Journal

Neuroscience

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Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

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Isolation and Culture of Primary Schwann Cells from a Human Dermis

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Learn to isolate and culture neural crest stem cells from human hair follicles using enzymatic digestion and flow cytometry. This protocol enables researchers to obtain CD271+ HNK1+ cells for regenerative medicine studies.

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

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