This work is funded by NIH/ NCI grant R01CA-118916

1. Extract Epithelial Stem Cells from Human Skin
<ol>
<li>Before starting the procedure of isolating epithelial stem cells one needs to prepare the respective medias and reagents (see table1).</li>
<li>Fresh adult human scalp skin from facelift procedures or punch biopsy is collected, then incubate in DMEM / 10% FBS / Dispase (4 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. Select follicles at telogen stage based on their morphology under a dissecting microscope, cut out the bulge region (Figure 1A) transfer follicles into a 15 mL sterilized tube. Anagen follicles can also be used if cut at upper 1/3 of follicle to obtain hair follicle "bulge" region (Figure 1B).</li>
<li>Incubate the isolated follicle fragments in a mixture of 0.05% trypsin-EDTA (GIBCO) and Versene (0.53 mM EDTA 4Na, GIBCO) (1:1) for 15-20 min at room temperature with shaking periodically, add 4 mL DMEM + 10% FBS to stop reaction, spin down for 5 min at 800 rpm. Alternatively, follicle tissue fragment explants can be placed in culture without trypsin digest to culture cells from single follicles.</li>
<li>Discard supernatant carefully (save ~0.2-0.5 mL to avoid losing cells) and re-suspend in 1 mL KCM (keratinocyte medium), without EGF.</li>
</ol>
2. Culture Primary Epithelial Stem Cells
<ol>
<li>Before culturing the epithelial stem cells, mitomycin C-treated 3T3-J2 cells (feeder cells) should be prepared as following. 3T3-J2 cells are cultured in DMEM with 10% FBS before treatment. Remove culturing media and wash cell twice with PBS, treat cell with 15μg / mL mitomycin C in DMEM without serum for 2 hr at 37°C, 5% CO2. Remove the mitomycin C-containing DMEM and wash twice with PBS, the cells are ready to use. (Mitomycin C-treated 3T3-J2 cells can also be prepared previously and stored at -80°C. Thaw and seed the cells one day before culturing primary epithelial stem cells, incubate at 37°C, 5% CO2. 150,000-200,000 3T3-J2 cells per well of 6-well plate is recommended.</li>
<li>Seed isolated hair follicle stem cell from step 1.5 on mitomycin C-treated 3T3-J2 cells in KCM without EGF overnight at 37°C, 5% CO2, changed to EGF-containing KCM the next day. Cells are grown at 37°C in a humid atmosphere containing 5% CO2. All keratinocytes are fed with KCM containing EGF every 2 days and grown for 14-20 days. Check cell every day under microscope. Hair follicle stem cells will form colonies (Figure 2A). Hair follicle explants will form outgrowths after 10-14 days (Figure 2B).</li>
</ol>
3. Passage Epithelial Stem Cells
<ol>
<li>Wash cells with PBS once. Remove 3T3-J2 feeder cells by Versene (pre-warmed to RT) treatment. Incubate culture dished in Versene for 5 minutes, then gently shake and aspirate off feeder cells.</li>
<li>Wash hair follicle stem cells with either Versene or PBS one time.</li>
<li>Add pre-warmed (to 37°C, critical) trypsin-EDTA (2X) and incubate at 37°C for 7 minutes or longer if necessary for the hair follicle stem cell to detach. No longer than 15 minutes is best.</li>
<li>Add KCM w/o EGF to stop the trypsin, gently pipette up and down to disperse cells, spin down at 200g, 5 minutes.</li>
<li>Re-suspend cells with KCM without EGF, Count and re-plate on mitomycin C-treated 3T3-J2 cells layer.</li>
</ol>
4. Immortalize Epithelial Stem Cells
Primary cells, even adult stem cells, reach senescence after serial passage and months in culture. Immortalization of primary stem cells is an effective way to relieve this problem.
<ol>
<li>Plate primary epithelial stem cells (~350,000 cells/well of 6 well plate) at passage 1 on feeder layer (3T3-J2) and culture for 2 days.</li>
<li>Culture PA317 LXSN 16E6E7 cell line one day after seeding the primary epithelial stem cells with DMEM containing 10% FBS. (This line produces the amphotropic retrovirus LXSN16E6E7 which encodes the HPV16 E6 and E7 open reading frames, and which can be used to stably infect and immortalize many cell types).</li>
<li>Treat PA317 LXSN 16E6E7 cell line with mitomycin C for 2 hours (Same protocol with 3T3-J2 cells). Add mitomycin C treated PA317 LXSN 16E6E7 cell line to the primary epithelial stem cells, co-culture for 6 days in KCM with EGF. Renew fresh KCM with EGF media every 2 days.</li>
<li>Remove 3T3-J2 and PA317 LXSN 16E6E7 cell with Versene. Add mitomycin-C treated 3T3-J2 NHP cells (neomycin, hygromycin, puromycin resistant, ~200,000 cells per well) to the epithelial stem cells. Cells are selected under 0.2 mg/ mL of G418 (Gibco) for additional 6 days. Renew fresh G418 containing KCM with EGF media every 2 days. 
Surviving epithelial stem cells will be immortalized stem cells. A well of primary epithelial stem cells which are not co-cultured with PA317 LXSN 16E6E7 cell should be set as a control for G418 selection, all the cells in this well should be killed by G418 after 6 days of selection.</li>
</ol>
5. Representative Results
Early passage of skin epithelial stem cells and immortalized epithelial stem cells form tight colonies consisting of small keratinocytes (Figure 2A) surrounded by feeder cells. If cultured in serum free media with defined supplements, without a feeder layer, the stem cells will disperse and not form tight colonies (they grow as single cells and small clusters). Immortalized epithelial stem cells maintain a stable phenotype for &gt;12 months of continuous passage, but tended to form tighter colonies than the primary cells. They do not form anchorage independent colonies in soft agar assays, indicating that these immortalized cells do not possess the characteristics of cancerous cells. Both primary and immortalized epithelial stem cells express hair follicle stem cell marker cytokeratin 15 (Figure 3A), and are able to differentiate into epidermal, hair follicle and sebaceous lineages (Figure 3B-D).
<img src="/files/ftp_upload/2561/2561fig1.jpg" alt="Hair follicle structure diagram; anagen, telogen phases; microscopic examination." data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Plucked hairs. After plucking from dispase-treated skin, hair follicles appear as either telogen club hairs (A) with a ball of cells surrounding the bottom of the hair, or anagen hairs (B) with a sleeve of epithelium surrounding the length of the hair. The telogen bulge region forms a distinct morphologic region to micro-dissect, while the anagen bulge is less distinct. The anagen bulb contains matrix keratinocytes, representing transit-amplifying cells that can also be isolated and cultured.
<img src="/files/ftp_upload/2561/2561fig2.jpg" alt="Embryo transfer process; microscope image showing cell injection technique in biology research." data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Stem cells cultures. (A) Epithelial stem cells from skin form tight epithelial colonies (designated by E) when cultured in KCM with a feeder layer of cells (designated by F). (B) Hair follicle explants give rise to epithelial outgrowths. The telogen bulge region (designated by T) is attached to the dish and surrounded epithelial cell colony along with feeder cells.
<img src="/files/ftp_upload/2561/2561fig3.jpg" alt="Fluorescence microscopy, cell staining, cellular structures; histological and immunofluorescence analysis." data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Stem cell differentiation. (A) Stem cells colonies maintain expression of epithelial markers such as cytokeratin 15. (B) Skin epithelial stem cells can be differentiated along the hair follicle lineage as determined by K6Hf expression. (C) The cells can be cultured on de-epidermalized dermis or other matrices at the air-liquid interface and will form a stratified epidermis with cornified layer, granular layer and spinous layer. (D) Stem cells can be induced along the sebaceous lineage with Oil Red positive globules.

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No conflicts of interest declared.

The cell extraction and culture methods described are surprisingly facile and reproducible. We have generated epithelial stem cell cultures from dozens of individuals across a broad age range, including patients with inherited skin defects18. It is best to begin the process on the day of tissue harvest, however cells will remain viable in media on ice for several days, facilitating overnight shipping if needed. Discarded facelift skin yields hundreds of viable follicles for cell extraction as single cell suspensions. For more limited tissue, such as from a punch biopsy of the scalp, explant cultures may be more effective, since there is less chance for cell loss in trypsinization and centrifugation. For explant cultures, the most important aspect is attachment of the tissue fragment to the dish. The addition of small amounts of media just covering the tissue and minimal manipulation is recommended until outgrowths appear in 10-14 days.
Adult epithelial stem cells from the skin could have a broad application. Cultured skin cells are already in clinical application for burn patients and chronic wounds. The challenge now has become creating a better skin equivalent with sweat glands and hair. The potential utilization of skin stem cells for hair regeneration is exciting possibility.
As mentioned previously, the ability to induce a primitive pluripotent state in adult cells represents a significant advancement for potential cell based therapies without the utilization of embryonic stem cells 16,17. However, the process is inefficient and potentially oncogenic, since viral vectors are used to induce expression of stem cell genes. If one starts with an adult stem cell population, it may greatly increase the efficiency and obviate the need for stable viral transfections to induce pluripotency. Transplantation of bone marrow stem cells has been utilized as a cell-based approach to treat severe skin diseases such as epidermolysis bullosa.19,20 In addition, correction of the collagen 7 defect in epidermolysis bullosa has been achieved in patient keratinocytes in vitro.21 Isolation of epithelial stem cells from a skin biopsy may provide resources to expand on this earlier work and develop individualized cell therapies using autologous cells.
It is interesting that the hair follicle bulge region is also the site of other adult stem cell / progenitor cell pools. Xu et al.22 has identified multipotent mesenchymal stem cells from this region. Melanocyte precursors also reside in the hair bulge23. Thus, the hair follicle isolation method presented here can easily be adapted to isolate these other cell types for further experimentation.

<table><tbody><tr><td>DMEM</td><td>GIBCO, by Life Technologies</td><td>11995</td><td></td></tr><tr><td>Hams F12</td><td>GIBCO, by Life Technologies</td><td>11765</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>GIBCO, by Life Technologies</td><td>16000</td><td></td></tr><tr><td>Insulin</td><td>GIBCO, by Life Technologies</td><td>12585</td><td></td></tr><tr><td>T3</td><td>Sigma-Aldrich</td><td>T-2752</td><td></td></tr><tr><td>Transferrin</td><td>Roche Group</td><td>10652202001</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich</td><td>H-4001</td><td></td></tr><tr><td>Cholera Toxin</td><td>Sigma-Aldrich</td><td>C8052</td><td></td></tr><tr><td>Epidermal growth factor (EGF)</td><td>Sigma-Aldrich</td><td>E-9644</td><td></td></tr><tr><td>Adenine</td><td>Sigma-Aldrich</td><td>A9795</td><td></td></tr><tr><td>Trypsin(10X)</td><td>GIBCO, by Life Technologies</td><td>15090</td><td></td></tr><tr><td>VERSENE</td><td>GIBCO, by Life Technologies</td><td>15040</td><td></td></tr><tr><td>G418 Sulfate</td><td>Cellgro</td><td>30-234-CR</td><td></td></tr><tr><td>Hanks&amp;rsquo; Balanced Salt solution</td><td>Sigma-Aldrich</td><td>H6648</td><td></td></tr><tr><td>1X PBS</td><td>Cellgro</td><td>21-040-CV</td><td></td></tr><tr><td>Mitomycin C</td><td>Roche Group</td><td>10107409001</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Invitrogen</td><td>15140122</td><td></td></tr><tr><td>Dispase</td><td>Invitrogen</td><td>17105</td><td></td></tr><tr><td>Crystal Violet</td><td>Fisher Scientific</td><td>C581-25</td><td></td></tr><tr><td colspan="4">&lt;p class=&quot;jove_step&quot;&gt;Keratinocyte media (KCM)&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;[DMEM and Ham s F12 (GIBCO, 3:1), adenine (Sigma, 180 mM), 10% fetal bovine serum (GIBCO), cholera toxin (ICN, 0.1 nM), penicillin/streptomycin (GIBCO, 100 U/ml and 100 mg/ml, respectively), hydrocortisone (Sigma, 0.4 mg/ml, 1.1 mM), T/T3 (transferrin, GIBCO, 5 &amp;#x03BC;g/ml, 649 nM; and triiodo-l-thyronine, Sigma, 2 nM), insulin (Sigma, 5 mg/ml, 862 nM), and EGF (Sigma, 10 ng/ml, 1.6 nM), pH 7.2]&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;[DMEM and Ham s F12 (GIBCO, 3:1), adenine (Sigma, 180 mM), 10% fetal bovine serum (GIBCO), cholera toxin (ICN, 0.1 nM), penicillin/streptomycin (GIBCO, 100 U/ml and 100 mg/ml, respectively), hydrocortisone (Sigma, 0.4 mg/ml, 1.1 mM), T/T3 (transferrin, GIBCO, 5 &amp;#x03BC;g/ml, 649 nM; and triiodo-l-thyronine, Sigma, 2 nM), insulin (Sigma, 5 mg/ml, 862 nM), and EGF (Sigma, 10 ng/ml, 1.6 nM), pH 7.2]&lt;/p&gt;</td></tr></tbody></table>

isolation and culture of adult epithelial stem cells from human skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

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Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

isolation-and-culture-of-adult-epithelial-stem-cells-from-human-skin

Nanyang Technological University

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28.3K Views.  University of Massachusetts Medical School. Een snelle, robuuste manier van isoleren levensvatbare volwassen epitheliale stamcellen uit menselijke huid is beschreven. De methode maakt gebruik van enzymatische afbraak van de huid collageen matrix, gevolgd door het plukken van de haarfollikels en isolatie van single cell suspensies of weefsel fragmenten voor celkweek.

Video: Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

Developmental Biology

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

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. ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in&#160;vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrin&#945;6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human ...

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 ...

Bioengineering

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

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