Following up on the previous article, Dr. Anastasakis explains the challenges associated with stem cells and hair follicle cloning in the treatment of Androgenetic Alopecia.
The small size and large number of hair follicles — along with the fact that they serve a non-essential function for survival — have led to the mistaken belief that their study involves a relatively simple structure. However, this perceived simplicity is highly misleading.
In reality, some of the most significant discoveries regarding epithelial–mesenchymal interactions and post-embryonic organ development have used the hair follicle as a model system. The hair follicle represents an attractive target in biomedical engineering because it is a readily accessible, complex, multicellular mini-organ that exhibits the same biological complexity seen in much larger, vital organs.
The scientific questions and barriers that must be overcome to engineer a functional hair follicle are no less formidable than those faced by researchers working to develop other tissues and organs.
Challenges and Theoretical Requirements for Hair Follicle Cloning
In order for stem cells to be successfully used to generate de novo hair follicles in animal models — and eventually in humans — two major bottlenecks must first be overcome:
The first challenge involves understanding the molecular signals that govern cell fate determination. In hair follicles, molecular signaling is particularly complex, as it requires consideration of cell types originating from two distinct germ layers.
The second challenge is achieving a clearer understanding of how tissue architecture is formed — specifically, how to arrange, proportion, and orient each cellular component correctly. We must learn how the number, size, and spatial distribution of hair follicles are consistently organized and replicated in mammals.
Overcoming these obstacles demands a deep understanding and integration of multiple biological processes and disciplines, including:
- Stem cell biology
- Epithelial–mesenchymal cell interactions
- Cell adhesion mechanisms
- Cytoskeleton and intermediate filament formation
- Regulation of stem cell formation and differentiation
- Cellular junctions, tissue organization, and structural compartmentalization
- Cell motility
- Intercellular communication
- Programmed cell death (apoptosis)
- Hormonal sensitivity at the cellular level
- Neurochemical–immune system interactions
- Cellular pigmentation
Bringing together knowledge from all these areas of biology has, as expected, proven to be extraordinarily complex and challenging to this day.
Studying the Fundamental Principles of Biology
The skin appendages of ectodermal origin in various animal species — including teeth, feathers, hair, glands, and scales — differ in shape, size, and function. However, they all originate from the same foundational biological mechanism: the interaction between epithelial and mesenchymal cells.
Although the bulge region appears to host the only progenitor epithelial cells in the lower portion of the hair follicle, the capacity for follicle formation is not exclusive to these bulge cells. Epidermal cells within the follicle retain some ability to generate new follicles, both in situ and after isolation and culture.
However, the follicle-inducing potential of bulge-derived cells appears to be the highest, suggesting that initiating therapies with a pure population of bulge stem cells could enhance the efficacy of cellular treatments. Still, these are not the only cells with inductive capabilities. In adult follicles, dermal stem cells also exhibit inductive properties.
In one of the earliest studies on de novo follicle generation (1966), Oliver demonstrated that if two-thirds of the dermal papilla or the upper two-thirds of the follicle remained intact, a new dermal papilla would regenerate — suggesting that dermal stem cells contributed to this reformation process.
The inductive potential of dermal stem cells has been confirmed in multiple studies where hair follicles were surgically amputated at different levels, and full regeneration occurred — as long as the damage stayed within specific limits. Altogether, these observations indicate that dermal stem cells possess trichogenic potential, much like the dermal papilla itself.
In a landmark study — though not yet replicated — Reynolds et al. implanted only the epithelial sheaths of human scalp hair follicles into a woman’s arm and reported the formation of new hair follicles. Their heterologous transplantation experiment suggested that dermal stem cells and the dermal papilla may evade immune rejection, and that these mesenchymal structures might enjoy a degree of “immune privilege,” even if they do not constitute a fully immune-privileged tissue.
Further studies have shown that epithelia, cultured epithelial cells, corneal epithelium, and amniotic fluid cells also possess the potential to generate hair follicles when exposed to inductive dermal cells. In fact, a variety of stem cell types — including embryonic, neural, and bone marrow–derived mesenchymal stem cells — have demonstrated the ability to form skin and hair follicles when introduced into blastocysts. This raises the intriguing possibility that other types of stem cells, when combined with appropriate inductive cells, could also lead to follicle formation — a possibility that remains to be further explored.
Trichogenic Cell Assays
Despite the variety of trichogenic cell assays described in the literature, all rely on the same core principle: combining epithelial responder cells with mesenchymal inducer cells within a suitable environment.
Epidermal stem cells can originate from multiple sources — embryonic stem cells, cultured embryonic stem cells or cell lines, follicular epidermal stem cells, bulge-derived stem cells, or even bone marrow stem cells. The trichogenic potential of dermal papilla cells (DPCs) in postnatal follicles has been experimentally demonstrated in landmark follicle culture studies by Jahoda et al., using vibrissae follicles from adult rats.
Numerous animal models for studying hair follicle regeneration have also been reported to date. As early as the 1960s, DPCs were identified by independent researchers as mesodermal components capable of inducing hair follicle formation. Since then, various trichogenic assays have been developed.
In general, each trichogenic assay can be classified based on:
- The origin of the experimental cell sample (e.g., neonatal dermis, postnatal DPCs, dermal stem cells, etc.)
- The condition of the dermal cell population being tested (e.g., cell clusters vs. isolated cells, cultured vs. uncultured, etc.)
- The epithelial counterparts used in the combination (e.g., amputated follicles, full-thickness epidermis, epithelial fragments, or isolated keratinocytes)
- The microenvironment of the reconstructed sample
- In vivo: e.g., subcutaneous space or under the renal capsule
- In vitro: e.g., liquid culture or 3D scaffold-supported culture
- The evaluation parameters used for efficacy testing (e.g., hair shaft elongation, partial or full follicle reconstruction, partial or complete folliculoneogenesis)
Below is a summary of current trichogenic cell assay techniques:
- Wound Assay: In this method, a small skin incision is created, into which freshly cultured DPCs are introduced using forceps or injection, just beneath the epidermis. This technique was successfully used by Jahoda et al. to achieve folliculoneogenesis.
- Chamber Assay: Originally developed by Lichti et al. and Weinberg et al., this method involves suspending freshly isolated or cultured DPCs together with neonatal epidermal cells and transplanting them into a round silicone chamber placed on the dorsal surface of hairless (nude) mice. Since hair shaft growth is visible to the naked eye, this assay has been widely used to confirm the follicle-inducing capabilities of epithelial and/or mesenchymal cells.
- Sandwich Assay: In this technique, DPCs are layered between enzymatically separated dermal and epidermal fractions, and the entire construct is transplanted either subcutaneously or under the renal capsule.
- Flap Assay: A recent modification of the sandwich assay, this technique uses embryonic mouse epidermis placed on a temporarily lifted dermal flap. Using this method, cultured human DPCs — even after eight passages — have demonstrated the ability to induce the formation of reconstructed hair follicles.
- Hair Patch Assay: A mixture of epithelial cells and DPCs is injected subcutaneously or under the renal capsule.
- Hair Patch Assay Variant: In a variation of the above, employed by at least two independent research groups, recently isolated bulge region cells from adult mice were combined with neonatal dermal cells. When this mixture was injected subcutaneously into immunodeficient mice, new hair follicles formed — highlighting the follicle-inducing potential of adult bulge-derived cells in the right inductive environment.
Growth Factors and Stem Cells
Several growth factors are involved in the molecular signaling between cells participating in hair follicle morphogenesis. IGF-1 and FGF-7 have been proposed as mediators that initiate signaling pathways from dermal papilla cells (DPCs) toward the follicular epithelial cells.
Wnt signaling has been shown to sustain the hair-inducing activity of DPCs. TGF-β2 is expressed in DPCs but not in dermal fibroblasts. Alkaline phosphatase activity is maintained in DPCs, while versican expression and alkaline phosphatase activity gradually decline during DPC expansion.
At each morphogenetic stage, these molecules are responsible for reciprocal signaling between the epithelial and mesenchymal components of the hair follicle. However, in the context of hair follicle neogenesis, it remains unclear which specific signaling molecules are responsible for inducing hair growth from transplanted DPCs. To date, certain signaling factors — such as bone morphogenetic protein (BMP)-6 — have been shown to enhance folliculogenesis in murine models. Wnt3a signaling from epithelial cells is also essential for maintaining the inductive capacity of DPCs and for initiating follicle formation.
These factors have been identified as candidate regulators of hair follicle morphogenesis through advanced transgenic approaches, including in vivo knockout and overexpression models. However, in humans, the practical and ethical challenges of applying transgenic techniques have limited studies aimed at clarifying gene-specific functions in vivo.
Despite promising findings, there is still no reliable biological marker for assessing the hair-inductive potential of human DPCs. As a result, in vivo animal models remain the only viable method for verifying the inductive capability of human DPCs at this time. Therefore, although several molecular markers have been reported to be specifically expressed in human DPCs, their functional roles remain to be fully elucidated.
Trichogenic Properties of Adipose Tissue–Derived Stem Cells (ADSCs)
In the context of hair follicle biology, several studies have demonstrated that growth factors from the follicular microenvironment can stimulate hair follicle development both ex vivo and in vivo in animal models. Some researchers have identified secreted factors in adipose tissue-derived stem cells (ADSCs) using ELISA and proteomic analysis, including PDGF, KGF, HGF, VEGF, and fibronectin — all of which are documented to promote hair follicle growth.
ADSCs exhibit multilineage plasticity and share several characteristics with bone marrow-derived mesenchymal stem cells (MSCs).
Moreover, ADSCs secrete cytokines with beneficial paracrine effects on surrounding cells and tissues. Recently, this paracrine activity has emerged as one of the most therapeutically relevant features of ADSCs. A recent study by Park et al. concluded that ADSCs may promote hair follicle development through a paracrine mechanism that is enhanced under hypoxic conditions.
In a recent in vitro study by Won et al., human ADSCs were isolated via liposuction, cultured according to standard protocols, and then co-cultured with human DPCs and immortalized keratinocytes. Although this technique successfully enhanced cellular proliferation, the results were inferior to those of the control group treated with 1 mM Minoxidil.
To date, it remains unclear whether certain secreted factors from ADSCs possess greater or lesser therapeutic potential for stimulating hair follicle development. Available data suggest that ADSCs promote hair growth primarily by enhancing the proliferation of DPCs — and potentially epithelial cells — through cell cycle modulation and activation of the anagen phase.
Therefore, while the rational use of ADSCs presents a promising approach for enhancing hair follicle regeneration, it cannot yet be considered superior to established pharmacological treatments such as Minoxidil, unless future results demonstrate significantly greater efficacy.
Challenges That Must Be Addressed for Human Hair Follicle Cloning
As of 2012, all experiments investigating the potential of hair follicle stem cells in hair follicle neogenesis have been conducted using rodent hair follicles, with perhaps the most significant being the landmark study by Jahoda et al., in which amputated human hair follicles transplanted into immunodeficient mice successfully generated functional hair follicles.
However, there are substantial differences between human hair follicles—particularly those of the scalp—and rodent follicles. The cellular and molecular features of human follicular stem cells are likely to differ greatly from those of rodents, as the anatomical characteristics of the follicles vary significantly: human scalp follicles can reach lengths of up to 5 mm, whereas mouse follicles measure only about 1 mm.
Moreover, although vibrissae follicles (whisker follicles), which are frequently used in research, are larger and therefore superficially more similar to human follicles, they are structurally atypical and behaviorally distinct, particularly in terms of their life cycle dynamics, making their use as models for human follicles problematic.
While rodent models have proven effective for advancing our understanding of human biology in general, their findings require confirmation in human systems. Despite providing insight into the biology of human hair follicles, almost all in vivo animal models used in follicle neogenesis research are unreliable and unpredictable, primarily due to technical challenges.
For example, in a model where human dermal papilla cells (hDPCs) were implanted subcutaneously into the ear pinna of rats via small skin incisions, it proved impossible to distinguish between native and newly regenerated hairs, as the original ear hairs vary greatly in size. Additionally, these models require extended timeframes to evaluate folliculogenic potential, and none are clinically practical for therapeutic use.
Some culture techniques result in follicle development beneath the skin, while others are too invasive or complex to be feasible treatments for a non–life-threatening condition such as alopecia areata (AA). In other studies, hDPCs were injected intradermally into mouse skin using syringes, but this approach has yet to yield successful human follicle formation.
To date, several key unresolved issues have hindered the success of these experiments:
- Although immunodeficient mice have been used as hosts, they may still be inadequate for allogeneic transplantation of human hDPCs. These models often exhibit gradual replacement of transplanted hDPCs by host epidermal cells.
- hDPCs lose their inductive capacity during in vitro expansion, and the molecular mechanisms responsible for this loss of trichogenic potential remain poorly understood.
- Human epidermal components, typically cultured keratinocytes, often fail to maintain their differentiation potential, possibly due to insufficient numbers of follicular stem cells. Research on epidermal stem cells suggests significant inter-species differences in the properties, behavior, and differentiation capacity of these cells. However, it’s difficult to determine how these differences affect follicle neogenesis in humans, as mesenchymal inductivity and epidermal differentiation are highly interdependent processes.
- Additionally, the difficulty in isolating sufficient numbers of both inductive hDPCs and undifferentiated epidermal stem cells presents a major limitation. Zheng et al. successfully achieved in vitro folliculogenesis via intradermal injection of neonatal epithelial and mesenchymal cells from mice. They found that the epithelial-to-dermal cell ratio influences follicle-forming ability. Their estimates suggest that approximately 5,000 dermal cells and 2,500 epidermal cells are required to generate a single hair follicle, making the process extremely resource-intensive.
Furthermore, human hair follicles do not grow synchronously, and the anagen phase in humans can last several years. Thus, the colony-forming efficiency (CFE) of individual anagen follicles can vary dramatically. For example, a follicle in anagen for only one month may yield a significantly higher number of colonies than one in anagen for 40 months. However, Roh et al. recently discovered a method to minimize follicular cycle variability in CFE by isolating telogen follicles with a well-defined bulge region.
The Potential Role of Stem Cells in Oncogenesis
Based on the premise that epithelial stem cells have a lifespan at least as long as that of the organism, it is believed that they are susceptible to cumulative genetic mutations (genetic hits), which may eventually lead to tumor formation. A substantial body of data from murine experiments suggests that many skin tumors originate from hair follicles and their resident stem cells.
Keratinocyte stem cells appear to be primary targets of carcinogenic agents. Given their slow proliferation rate, these stem cells are thought to retain carcinogens for extended periods, making them more vulnerable to tumorigenesis.
Specifically, there is considerable evidence that basal cell carcinomas (BCCs) may arise from hair follicle stem cells. BCCs have been shown to express bcl-2, a marker of permanent follicular compartments, including the bulge region.
Since BCCs are slow-growing tumors composed of poorly differentiated cells capable of differentiating into various appendageal structures, it has been proposed that the cell of origin is a multipotent, slow-cycling stem cell with high proliferative potential.
Additionally, overexpression of Sonic Hedgehog (Shh) in the mouse epidermis induces BCC-like tumors through follicular budding, while having minimal effects on the interfollicular epidermis. This finding supports the hypothesis that BCCs originate from the hair follicle.
Trichoepithelioma (TE) is a benign follicular tumor that shares clinical and histological features with BCC. Both tumor types may coexist in the same patient, and it has been proposed that they may derive from a common progenitor cell type within the follicle. Findings indicating that sporadic TEs and BCCs harbor mutations in the Patched (ptc) gene provide strong evidence of a shared oncogenic mechanism and a follicular origin.
The normal expression of the ptc gene in developing hair follicles, along with the presence of ptc mutations in sporadic TEs as well as both hereditary and sporadic BCCs, supports the hypothesis that these tumors originate from a common cell type within the hair follicle. Further investigation of TEs, BCCs, and other follicular tumors using additional markers for follicular stem cells may yield valuable insights into the role of stem cells in tumor development.
Until such mechanisms are fully understood, the use of stem cells for hair follicle neogenesis may not be entirely safe in the long term.
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