Thank you for visiting nature. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. The hair follicle HF is a highly conserved sensory organ associated with the immune response against pathogens, thermoregulation, sebum production, angiogenesis, neurogenesis and wound healing.
Although recent advances in lineage-tracing techniques and the ability to profile gene expression in small populations of cells have increased the understanding of how stem cells operate during hair growth and regeneration, the construction of functional follicles with cycling activity is still a great challenge for the hair research field and for translational and clinical applications.
Given that hair formation and cycling rely on tightly coordinated epithelial—mesenchymal interactions, we thus review potential cell sources with HF-inducive capacities and summarize current bioengineering strategies for HF regeneration with functional restoration.
Hair follicles HFs are a major skin appendage originating from the ectoderm. As a stem cell repository and a hair shaft factory, the HF contributes to remodelling its cutaneous microenvironment, including skin innervation and vasculature.
In addition, hair in human society greatly affects the quality of life, attractiveness and self-esteem. However, destructive inflammation with various aetiologies and the subsequent replacement of fibres can involve the permanent loss of HFs, which impairs inherent skin function and, especially, psychological well-being. Thus, HF regeneration is in ever-increasing demand and has promising market prospects. HF morphogenesis and regeneration were shown to be dependent on the intensive cooperation of epithelial epidermal stem cell [Epi-SCs] and hair-inducive mesenchymal dermal papilla [DP] components, also called epithelial—mesenchymal interaction EMI.
EMI is a prerequisite for functional HF formation, regeneration and cycling, mainly through paracrine mechanisms, 2 and has become the theoretical basis of tissue engineering for HF regeneration. HFs are also a dynamic mini-organ, and their most notable feature is hair cycling Fig. Thus, the achievement of hair cycling regeneration is important for functional HF regeneration. Although hair transplantation has been widely applied, transplanted hair is not maintained in the long term.
Current techniques could make it possible to obtain potential cells in vitro Fig. In addition, the optimization of the culture system also contributes to preserving the HF-inducive ability of potential cells. Based on these findings, we are able to regenerate functional HFs by the transplantation of potential cell mixtures, HF organoid construction in vitro, reprogramming induction and the establishment of a drug delivery system Fig.
Here, we will review the potential cell sources and tissue engineering techniques that contribute to HF regeneration. In addition, the limitations and future of functional HF regeneration are summarized. The transformation of fibroblasts into DPCs via lineage reprogramming. Optimization of the in vitro system to preserve the HF-inducive potential and the transplantation of cell-based biomaterials or HF organoids in vivo to regenerate HFs.
A mixture of SKPs and epidermal stem cells or bioactive peptides was embedded in the hydrogel to regenerate HFs three-dimensionally. Past studies reported that DPCs isolated from rat and guinea pig vibrissae, as well as humans, could also induce HF formation when implanted into recipient non-hairy skin, 9 , 10 which indicates that DPCs could reprogramme non-hairy epidermis to a follicular fate. Subsequently, DPCs, either fresh or after tissue culture expansion, could also reproduce new HFs if placed in proximity to the epithelium.
It has been reported that sphere formation increases the ability of cultured human DPCs to induce HF from mouse epidermal cells, 32 in which glucose metabolism 33 and epigenetics 34 may be important regulators.
JAK inhibitor regulates the activation of key HF populations, such as the hair germ, and improves the inductive potential of DPCs by controlling a molecular signature enriched in intact, fully inducive DPs. DSCs are stem cells located in the dermis of the skin. Based on distinct phenotypic properties and different cultural environments, DSCs can be divided into dermal fibroblasts and SKPs.
SKPs have the capacity to differentiate in vitro and in vivo into multiple lineages of different progeny. Trichostatin A, a potent and specific inhibitor of a histone deacetylase, could restore the HF-inducive capacity of SKPs by markedly alleviating culture expansion-induced SKP senescence, increasing the expression and activity of ALP and elevating the acetylation level of histone H3.
They are divided into quiescent and primed HFSCs. For instance, relatively quiescent HFSCs in the follicular bulge region can serve as a reservoir for transient amplifying cells that are able to produce various cell types during HF regeneration.
Recent studies have reported that basal keratinocytes also have the capacity to facilitate HF regeneration under induction. The differentiation of stem cells into adult cells in response to defined factors is an important application of cell reprogramming. Induced pluripotent stem cells iPSCs have similar characteristics to embryonic stem cells in terms of morphology, self-renewal and differentiation capacity.
They are not only free from ethical issues but also able to be propagated as autologous cells, which can avoid the complication of immune rejection. Thus, reprogramming of iPSCs into potential cells could be an approach to providing cell sources for HF regeneration. Lineage reprogramming is direct cellular reprogramming, which means that targeted cells could bypass the stem cell stage and convert directly to potential cells. Unlike traditional concepts regarding the epigenetic stability of differentiated cells, direct lineage reprogramming can transform one specialized cell type into another using defined factors, which is a more efficient and promising approach for producing functional cells.
With progress in HF developmental biology and cellular reprogramming techniques, several cells with the potential for HF regeneration have been identified. These results have greatly expanded the seed cell bank for HF regeneration and solved the problem of the lack of a cell source. However, the problems these cell sources have in common are that their potential to regenerate HF fails to be maintained during long-term culture in vitro.
In addition, HFSCs are few in number and extremely difficult to obtain, and SKPs in vitro senesce soon when isolated from their physiological environments. The optimization of the culture system in vitro and the improvement of reprogramming efficiency are challenges for HF regeneration. Therefore, we believe that the construction of 3D culture systems that simulate the in vivo environment may provide an alternative approach, similar to hydrogel scaffold-based cell culture, which contributes to maintaining cell proliferation and growth as well as the potential to regenerate HFs.
In addition, chemical reprogramming, a new reprogramming technology, is characterized by high security and efficiency. After birth, mature and actively growing HFs eventually become anchored in the subcutis and then undergo hair cycling, periodically and spontaneously undergoing repetitive cycles of growth anagen , apoptosis-driven regression catagen and relative quiescence telogen.
Therefore, the HF is regarded as a dynamic mini-organ. The activation and maintenance of hair cycling is a prerequisite for the functional regeneration of HFs. HFSCs play an indispensable role in maintaining hair cycling.
The HFSC population remains largely quiescent during hair growth, but a subpopulation actively proliferates and promotes the production of the new hair shaft under the control of Axin2 expression. The mechanism underlying HFSC homeostasis and hair cycling regulation is a complex molecular controlling process Table 1 that is highly dependent on hormonal action.
The dynamic characteristics of HFs enable their sustainable and periodic regeneration. We think that the activation and maintenance of hair cycling are indispensable for achieving functional HF regeneration.
Past studies have not only led to a better understanding of the molecular mechanisms of hair cycling, which makes it more possible to find the key molecules therein, but also have revealed the complexity of HF dynamic characteristics. Therefore, it is still difficult to discover the key molecular event in hair cycling. Cell-based transplantation without biomaterials is a minimally invasive approach to in vivo HF regeneration.
Current cell transplantation mainly involves the transplantation of stem cells or a mixture of epidermal and dermal components. The injection of a mixture containing Epi-SCs and DPCs into nude mice could induce new HFs with the correct histological structures and form a multilayered stratified epidermis containing HF-like structures. A mixture of Epi-SCs and SKPs was grafted into excisional wounds in nude mice, and a bilayer structure resembling the epidermis and the dermis formed on the fifth day, followed by de novo HF.
More importantly, this experiment also demonstrates that the PI3K-Akt signalling pathway plays a crucial role in the interactions between Epi-SCs and SKPs and de novo HF regeneration, which may suggest potential therapeutic applications in enhancing hair regeneration.
In recent years, for the fully functional regeneration of ectodermal organs, a bioengineered organ germ has been developed by reproducing the embryonic processes of organogenesis, including bioengineered HFs. The bioengineered HFs not only properly connected to the host skin epithelium by intracutaneous transplantation and reproduced the stem cell niche and hair cycling but also autonomously connected with nerves and the arrector pili muscle at the permanent region and exhibited piloerection ability.
After intracutaneous transplantation, this bioengineered HF germ not only develops the correct structures and forms proper connections with surrounding host tissues such as the epidermis, arrector pili muscle and nerve fibres but also shows restored hair cycling and piloerection through the rearrangement of follicular stem cells and their niches, with fully functional hair organ regeneration.
Cellular reprogramming is not only a tool for tissue engineering to enrich potential cell sources for the regeneration of HF but also a participant in physiological de novo HF induction. Secreted proteins apolipoprotein-A1, galectin-1 and lumican from embryonic skin conferred upon non-hair fibroblasts the competency to regenerate HF via the activation of IGF and WNT signalling, thereby endowing non-HF skin with the ability to reproduce HFs, which suggests the involvement of cellular reprogramming.
Biomaterials are implantable, inactive materials that can replace or repair damaged tissue with high biocompatibility. Biomaterials can create a 3D environment for cell-to-cell interactions, simulating the function of cell niches to a certain extent, and they have been widely used in wound repair and tissue regeneration. It has been reported that DPCs retain HF inductivity best when cultured and transplanted as multicellular aggregates, and DP spheroids could form a structure similar to the natural intercellular organization in vivo.
Larger DP spheroids exhibit higher HF inductivity. The researchers developed a method that can be automated for mass production of DP spheroids with controllable size and cell number in a wide range, 93 although the average diameter of regenerated hair fibre did not significantly change with increasing size of the transplanted DP spheroids.
Likewise, the hanging-drop approach could also lead to a controllable 3D spheroid model for the scalable fabrication of inductive DP microtissues. That technique is based on surface tension and the interaction between surface tension and a gravity field that causes the convergence of liquid drops.
With the converged drops, DP spheroids could endow high-passaged DP microtissues with many similarities to primary DP. Subcutaneous implantation of these microtissues mixed with new-born mouse epidermal cells has achieved reproducible HF induction in the hypodermis of nude mice, and a large amount of extracellular matrix ECM components is found in the intercellular space within the DP microtissue, similar to an anagen DP.
Local injections of 3D DP-Exos exosomes could induce anagen from telogen and prolong anagen in mice. Moreover, DPC spheres treated with Exos could augment HF neogenesis when implanted with mouse epidermal cells, 95 which may be associated with high levels of miRp in Exos.
Owing to the security and tuneable thickness at the nanoscale, the nanogel could encapsulate a single cell by layer-by-layer LbL self-assembly and further form DPC spheroids by physical cross-linking on nanogel-coated cells. Remarkably, the vascularization of HF-bearing human skin constructs increases graft survival and enables efficient human hair growth in mice. It is likely that SKPs rely on special environments for their self-renewal and stable gene expression. In tissue engineering, scaffolds are created to mimic environments for stem cells to survive, differentiate and form functional tissue structures.
In this regard, scaffolds such as hydrogels and matrix could be candidates to support stem cells for organogenesis and regenerate HFs.
The transplantation of a combination of culture-expanded SKPs and neonatal epidermal cells into RADA-PRG hydrogel resulted in a significantly increased number of neogenic hairs compared to Matrigel and other peptide hydrogels.
This may be attributed to the similarity of the properties of these designer peptide nanofibres to those of ECM molecules. Normal human neonatal foreskin keratinocytes were induced to differentiate into several cellular components that compose normal HFs, with the expression of anagen-specific versican when grown on a collagen matrix embedded with TSC2-null fibroblast-like cells or with fibroblasts.
Drug delivery systems consist of molecules with pharmacological activity modified into advanced materials, which have been widely used in skin wound treatment. Currently, it has been reported that many drug delivery systems could promote wound healing as well as HF regeneration.
Newly developed multidomain peptide hydrogels have exhibited regenerative potential in a diabetic wound healing model, resulting in wound closure, accelerated HF regeneration and a greater average number of HFs at both the edge and the centre. The fibrous membrane can effectively release Qu and Cu ions to induce the proliferation, migration and differentiation of skin- and HF-related cells, and the Qu, Cu ions and Si ions released from the composite membrane revealed synergistic activity to stimulate HF regeneration and wound healing in burned skin.
WIHN is a regenerative phenomenon separate from physiological regeneration, as its cellular origin is not from the HFSCs in the bulge at the wound edge. In WIHN, a fully functional follicle can regenerate in the centre of a full-thickness wound with a large enough size, and the cellular origin of this process is similar to an embryonic process.
The neogenic follicles have similar functions to embryonic HFs, which also have a growth cycle. However, because of their controllability, targeted delivery, sustained release and even intellectuality, we think drug delivery systems hold great promise for HF regeneration in the future.
An organoid is defined as a 3D structure grown from organ-specific stem cell types. It can recapitulate key aspects of in vivo organs and avoid many of the disadvantages associated with cell lines. DP spheroids encapsulated by silk-gelatine hydrogel and HF keratinocytes as well as stem cells could be used to construct in vitro HF organoids.
These organoids show enhanced DPC-specific gene expression and ECM production, and their structural features and cell—cell interactions are similar to those of in vivo HFs. To date, in vitro skin derivation strategies have focused on first generating keratinocytes and fibroblasts from iPSCs in separate cultures and then combining the two types of cells to form a skin-like bilayer. A major challenge is how to realize the synchronous construction of its appendages. However, the new HFs entered catagen and degenerated during long-term culture.
Scalp-derived dermal progenitor cells mixed with foreskin-derived Epi-SCs at a ratio could aggregate in suspension to form a large number of HF organoids, and the dermal and epidermal cells self-assembled into distinct epidermal and dermal compartments.
The addition of recombinant WNT3a protein to the medium enhanced the formation of these aggregates, and the transplantation of these organoids in vivo achieved HF formation.
The transition from traditional culture to 3D culture constitutes excellent progress in HF regeneration. In 3D culture, regenerated HFs in vivo not only connect appropriately to surrounding host tissues but also undergo hair cycling activation. However, some problems are less thoroughly addressed. How long can the regenerated HFs last? Can the regenerated HFs go through full hair cycling? Will HF regeneration-induced hair be superior to transplanted hair? We think these are key questions and important challenges in functional HF regeneration.
Much progress has been made in the developmental biology and regeneration of HFs. Since it is an architecturally and functionally complex organ, the HF is much more difficult to regenerate or reconstruct than many other organs. Due to this limitation, HF regeneration is still far from clinical transformation. Both cell transplantation and organoid architecture lack the microenvironment of connective tissue, blood vessels and immune cells, which is still quite different from the physiological environment of normal tissues and organs.
Do they allow other essential cells to be recruited to the new follicle? If so, do the attracted cells have the ability to affect organogenesis overall? Ideal biomaterials need to be safe and nontoxic. Under normal metabolism in the body, they can be kept in a stable state without biological degeneration, and the metabolism or degradation products are harmless and easily metabolized. The combination of different technologies and methodologies will hopefully lead to new progress.
For example, the creation of transplantable HFs that closely mimic the structures and functions of native tissue may be accomplished by combining organoid technology with a drug delivery system. Depending on the controllable release of the relevant factors in hair cycling, such as WNT or BMP, hair cycling activation and maintenance of HF organs may be achieved. We also need to continue to optimize the in vitro culture systems of potential cells and look for more efficient reprogramming techniques, such as chemical reprogramming induced by small molecules or genetic reprogramming of genes delivered by biomaterials.
Finally, we want to reiterate that, based on existing work, it is worth considering whether the achievement of the activation and maintenance of hair cycling in regenerated HFs could be the heart of the next phase.
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A two-step mechanism for stem cell activation during hair regeneration. Cell Stem Cell 4 , — Dynamics between stem cells, niche, and progeny in the hair follicle. Using a systems biology approach, we also identified several master regulator MR genes of inductive DPC identity, which could potentially be used to achieve complete restoration of hair inductive transcriptional signature of DPCs 7.
Similarly, we recently reported that Jak inhibitors also directly restore hair inductivity in treated DPCs in culture In this study, we present an innovative biomimetic approach for effective generation of human HFs within HSCs by recapitulating the physiological 3D conformation of cells in the HF microenvironment. Further, vascularization of hair-follicle-bearing HSCs increases graft survival and enables efficient human hair growth in mice.
Our method represents a novel bioengineering strategy for feasible generation of hair-bearing HSCs entirely ex vivo from cultured human cells. Human DPC spheroids have the potential to induce de novo hair formation when placed in contact with human epidermis in mice 7 , In agreement with previous studies 14 , this observation indicated that simple engineered HSCs do not provide a physiological microenvironment sufficient to induce HF formation.
To control the spatial arrangement of cells in HSCs, we used 3D-printing technology and microfabricated plastic molds that contain HF-shaped extensions that are adjustable in diameter, length, and density Fig. These molds were used to create an array of microwells on a type I collagen gel containing dermal fibroblasts FBs Fig. Seeding DPCs over these microwells led to spontaneous aggregate formation in the base of the microwells overnight Fig.
This method allowed for precise control of DPC aggregate size by adjusting the diameter of the microwells Fig. These changes were not observed in FB aggregates Fig.
Patterning of collagen type-1 gel using 3D-printed molds allows for physiological arrangement of cells in the hair follicle. Hair follicle molds were designed a and 3D-printed b to have HF-like extensions and a 5-mm-deep cavity that allows the molds to float on collagen gel. To establish a physiological conformation of cells, we seeded keratinocytes KCs over the dermal constructs and allowed the cells to settle down and fill up the microwells, engulfing the DPC aggregates Fig. Reflecting the physiological proximity and conformation of epidermal and mesenchymal cells, KCs directly above the DPC aggregates showed differentiated morphology resembling KCs in the HF after a few days in culture Fig.
Culturing the 3D skin constructs for a week resulted in the differentiation of KCs into specific hair lineages, including Keratin-5 K5 an outer root sheath marker , AE13, AE15 and K71 inner root sheath markers , and K75 a hair medulla and companion layer marker Fig.
When the same experiments were performed with FB aggregates as a control, KCs remained in their undifferentiated state by retaining the expression of K5, and failure to express the hair lineage markers, AE13 or K75 Fig.
In these analyses, we did not observe differentiation of KCs into the sebocyte lineage as evident by the absence of Oil-red-O staining within the follicular structures Supplementary Figure 2.
Differentiation of human keratinocytes into specific hair follicle lineages in HSCs. Extending the culture period from 1 week to 3 weeks in vitro led to the elongation of the HFs down into the dermis and a better organization of the inner and outer root sheath layers Fig. Remarkably, in some of our constructs, we observed hair fibers protruding from the surface of the HSCs Fig.
Although at this stage, this process was relatively inefficient and was only observed approximately in one out of three constructs. However, to our knowledge, this is the first demonstration of the generation of human HFs in HSCs in an entirely ex vivo context. Therefore, to further enhance hair inductivity, we leveraged two MR genes, Lef-1 and Fli-1 , which we previously identified as the key regulators of the intact DPC gene signature 7. Interestingly, our previous systems biology approach showed that both spheroid culture of DPCs and Fli-1 overexpression restored very similar expression profiles 7.
In contrast, Lef-1 targeted genes that were not restored by spheroid culture, suggesting that Lef-1 overexpression and spheroid culture are complementary for restoration of the DPC hair inductive transcriptional signature. To test this hypothesis, we performed transient transfection of Lef-1 in cultured DPCs passage 3 Supplementary Figure 3 , and then formed spheroids with Lef-1 -transfected cells.
Gene distance matrix Fig. Skin constructs generated with DPCs overexpressing Lef-1 resulted in significant increase up to fold in the expression of the specific hair lineage genes, including the outer and inner root sheath K17, K71, K25 , and hair companion and medulla markers K75 , compared to the DPCs transfected with empty vector Fig. The fold differences in Fig. Fold difference values are based on the empty vector-transfected DPCs.
To extrinsically recapitulate the role of Lef-1, we treated the constructs with recombinant human Wnt10b and CHIR, a small molecule that activates Wnt-signaling through inhibition of GSK3. The exogenous expression of the other MR gene, Fli-1 , resulted in significant reprogramming of the gene expression in cultured DPCs Supplementary Figure 5a and upregulated several Fli-1 downstream network genes, such as collagen 5 COL5 and collagen 6 COL6 Supplementary Figure 5b , in agreement with our previous gene network analyses.
Overall, these data supported our previous predictions that Fli-1 overexpression and spheroid formation have an overlapping effect in DPC reprogramming, whereas Lef-1 overexpression complements aggregate formation and significantly improves the restoration of DPC hair inductivity.
We next examined the capability of our HSCs containing Lef-1 -transfected DPCs to grow human hair in vivo by grafting them onto immunodeficient nude mice. To more closely recapitulate the physiological hair density in human scalp 13 , we increased the HF density from 81 HF to HF per cm 2 Fig. The first set of experiments did not result in hair formation; instead, we observed substantial necrosis at the center of the grafts due to lack of host vascularization in the grafts Supplementary Figure 6 a, b , consistent with our previous work 5.
Vascularization of high hair-follicle-density HSCs for efficient engraftment. GFP-tagged HUVECs that were encapsulated in the dermal compartment with the fibroblasts closely surrounded the Kpositive cells in the hair follicle structures d and formed capillary-like networks after 3 days of culture e.
Immunofluorescent wholemount imaging of the constructs revealed that these capillary-like structures were in close proximity to the HFs Fig. Grafting the vascularized HSCs onto mice promoted host vascularization into the grafts Fig. GFP-tagged HUVECs formed more organized and elongated networks upon grafting, which were located in close proximity to the host red blood cells and newly formed host vessels of the mice that were labeled with mouse-specific rhodamine-conjugated Isolectin Griffonia simplicifolia GS - IB 4 Fig.
Four to five weeks after grafting our vascularized HSCs at a high follicle density of HF per cm 2 onto immunodeficient nude mice, we observed substantial hair growth in the grafts, whereas the HSCs prepared with FB aggregates did not induce hair formation Fig.
In the grafting experiments, we used ten mice per condition. Grafts from four out of these seven mice successfully generated human HFs, whereas none of the seven mice in the FB control experiment induced hair formation. Low magnification images of the human-specific nuclear staining and K14 stain appears in multiple layers due to tilted angle of the constructs clearly delineated the edges between the host and grafted skin, and most of the regions of the mouse skin lacked HFs Fig.
Our grafts have only a slightly thicker epidermis than the mouse, consistent with our grafting strategy and timing, in which we chose to not cornify the epidermis at high calcium conditions to avoid any interference with HF differentiation as opposed to epidermal differentiation prior to grafting. Therefore, further epidermalization and cornification seen in Fig.
Induction of human hair growth in immune-deficient nude mice. Bright-field microscopy of unpigmented terminal human hair f , engineered human hair in the grafts g , and unpigmented human vellus hair h showed morphological similarities between human hair and engineered hair. Microscopic bright-field images of the hair fibers showed that engineered hair Fig.
To confirm the human origin of the de novo HFs, we selectively isolated the RNA of the cells from the HFs within the grafts using laser capture microdissection Supplementary Figure 8 a, b and performed PCR using human vs.
The PCR analyses with both sets of primers confirmed that HFs in the grafts highly expressed the human-specific sequence, whereas the control whole mouse skin tissue only expressed mouse-specific genes Fig.
We also observed low levels of mouse-specific gene expression in the HFs, consistent with presence of some cells in the grafts that are negative for human-specific nuclear staining Fig. These cells may represent recruitment of mouse vasculature and infiltrating cells to the microenvironment of the HFs, since the levels of mouse-specific gene expression were much higher in the whole grafts compared to the laser captured HFs Fig.
Tissue engineering of human HFs has been a long-standing challenge and its progress has lagged behind other lab-grown tissues, such as vasculature 16 and intestinal epithelium This is mainly due to the lack of availability of a platform that can successfully recapitulate the microenvironmental cues required to maintain the requisite cell interactions for hair neogenesis.
This biomimetic approach led to KCs differentiation into specific hair lineages and allowed us to generate human HFs within HSCs in an entirely ex vivo context.
For instance, creating de novo adult HFs from cultured cells dates back to , where cultured rodent HF DPCs induced hair growth in recipient skin The same group was the first to achieve hair growth in reconstructed follicles in culture Similarly, two recent studies established the growth of mouse hair in vivo through encapsulation of mouse iPSCs 20 or mouse adult DPCs and epidermal cells 21 in hydrogels followed by transplantation of these in vitro-conditioned structures into mice.
Another recent study also demonstrated the spontaneous formation of HFs in embryoid bodies of mouse iPSCs Despite these successes, significant interspecies differences exist between human and mouse HFs not only in their hair cycles, stem cell characteristics, and hormonal dependencies 23 , but also in the hair inductive properties of their dermal cells.
One striking difference is that cultured rodent DPCs can self-aggregate when transplanted onto rodent skin 24 ; however, this aggregative behavior is not observed in human DPCs In a study from Toyoshima et al. However, to translate and scale such an approach into a clinical reality, these cells must be expanded in vitro for several passages, which causes a rapid loss of hair inductive gene signature in DPCs 7 , 8. Subsequently, other groups also reported the use of this method to induce HFs in mice, albeit inefficiently This will be addressed in the future by leveraging appropriate growth factors, small molecules or proliferation enhancers.
For example, for third degree burn patients that require full-thickness skin grafts, spatial control over HFs may facilitate recreating the variations in hair density at the site of transplantation e. Interestingly, Plikus et al. The presence of HFs in our HSCs may enable this function by promoting adipocyte differentiation in the wound bed, although this requires further investigation.
In addition, the formation of a scar or nonfunctional skin is typically observed in skin grafts due to poor viability and integration of HSCs 27 , Our ability to vascularize hair-bearing HSCs will significantly promote the viability of both the skin and hair tissues, constituting a groundbreaking innovation for regenerative skin therapies.
Robotic hair restoration surgery has revolutionized modern hair transplantation by allowing for performing repetitive maneuvers within high precision and speed Using our strategy, we typically generate 15 million DPCs passage 3 from a strip of donor tissue as small as 0. The improvements of these robotic systems to harvest hair follicular units groups of 1—4 hairs , as opposed to single hairs, yield significantly more hair growth per harvest Moreover, from a technical perspective, harvesting HFs with an obtuse angle, compared to 90 degrees, is more challenging and typically requires larger incisions In our 3D-printing approach, we used a starting HF angle of 90 degrees, which is then self-reorganized within the constructs above degrees after 3 weeks of culture or skin grafting.
However, for hair restoration applications, we envision that HFUs in our hair constructs can be harvested as soon as a week after their preparation, providing a more practical starting material to harvest HFs with a straight incision perpendicular to the epidermis.
Organ culture of HFs is still the current gold standard for drug testing on human HFs. This method has several limitations including its low-throughput, dependence on fresh, living HFs from human donors, difficult to standardize due to wide inter-individual variation in rates of hair growth due to different human donors , inability to capture the signaling from the dermis e.
Our ability to incorporate engineered HFs into HSCs represents the first major step to circumvent some of these limitations. We are currently able to make nine skin constructs containing HFs in a six-well-plate format starting from only one HF donor tissue, which is a substantial improvement on the throughput aspect of drug testing in the organotypic assay.
In addition, the contribution of the dermal compartment on the HFs can potentially be examined especially with the addition of other dermal components in the future such as immune cells. One of the immediate extensions of our approach will be the incorporation of melanocytes to generate pigmented HFs. We previously derived melanocytes from iPSCs, and showed their capability to transfer their melanin to neighboring keratinocytes Moreover, in our analyses we did not examine the capability of these engineered HFs to undergo the hair cycle, which relies on the presence and maintenance of a reservoir of stem cells in our HSCs.
The ability to maintain the holoclonal epidermal stem cells in a feeder-free environment over several passages, as well as within HSCs over long periods, could provide greater regenerative capacity to engineered HFs for translational purposes. Alternatively, incorporation of HF stem cells 35 , 36 or iPSC-derived folliculogenic epidermal cells 37 separately into HSCs could potentially establish the hair cycle.
However, adding a stem cell niche, such as the bulge region, containing HF stem cells into our constructs will require better resolution in the spatial control of cells, which is a current technical limitation of our method utilizing 3D-printed molds. In the future, 3D-bioprinting technology operating at a single cell resolution may permit the inclusion of other cell types, such as stem cells and melanocytes, to generate cycling and pigmented HFs.
A recent study revealed that hair organoid formation of mouse cells in vitro follows a different self-organization mechanism than what occurs during hair morphogenesis, despite resulting in successful hair growth in vivo Our novel biomimetic developmental tissue engineering strategy represents a crucial step forward in generating a truly functional human skin and marks a dramatic conceptual advance in regenerative medicine approaches to disorders of the skin and HF, as well as improving the outcome of severe skin injuries leading to disfiguring scars.
Adaptation of this new technology by hair researchers, hair restoration surgeons and the pharmaceutical and cosmetic industries will have overwhelming implications in the maintenance and regeneration of this complex human tissue.
DPCs were isolated from discarded scalp tissues from hair restoration surgery kindly provided by Dr. Culture medium was changed every other day for all cell types. All 3D-printed molds were designed and drawn using the computer-aided design CAD software, Solidworks. The constructs were maintained in low calcium epidermilization medium 39 submerged for 1—3 weeks.
After removal of the 3D-printed molds, HSCs were first left in EGM-2 medium kit supplemented with growth factors Lonza for 3 days for capillary formation. After a day of culture in a mixture of EGM-2 and the epidermilization medium, the constructs were either imaged or used for grafting experiments.
For immunostaining and histological staining, samples were cut in half and embedded in paraffin wax or cryopreserved in OCT solution. Wholemount tissue staining was performed similarly to the staining of the sections except that the tissues were incubated in the primary antibody for 3 days and the secondary antibody overnight.
The tissue was cleared by benzyl alcohol to benzyl benzoate mixture before imaging. Only one chamber was placed on each mouse. The chamber was hat shaped, with a hole in the top of the hat. The HSCs were placed into the chambers and maintained for 5 days by adding epidermalization medium everyday into the chamber.
Subsequently the chamber was removed and the HSCs were secured with four sutures 7—0 Nylon in a simple interrupted pattern around the edge of the graft. The mice were euthanized after 4—6 weeks for analyses.
DPCs at passage 3 were seeded in six-well plates at , cells per well and cultured overnight prior to transfection. Lipofectamine P transfection reagent was used to transfect the cells. Samples were processed at 30 million reads per sample. For the sequencing analysis, reads were aligned to the human reference genome hg Gene counts were calculated using HTSeq and were used as an input for differential gene expression analysis with DESeq version 1.
Gene expression data of the samples clustered in heatmaps and gene distance matrix were performed using unsupervised hierarchical gene clustering analyses. A null distribution for calculating the NES and its associated p value was found by label shuffling to randomize the gene rankings.
These randomized sets were then used to calculate null ESs over 10, iterations to generate a null distribution. The observed leading-edge ES was normalized to this null distribution, and a two-tailed p value was generated for this NES.
Fold changes were calculated using the delta-delta CT algorithm. Error bars were calculated based on SD across three technical replicates within three biological replicates. Primer sequences are available in Supplementary Table 1.
The captured tissue was immediately lysed and prepared for further PCR analyses. Since we used four different cell types in our engineered tissues and anticipated variabilities between different donors, we chose an adequate sample size by using at least three biological replicates and three technical replicates throughput the study. In particular, the experiments done for establishing HF differentiation in hair constructs in vitro were performed in triplicates using cells from the foreskin of three different donors biological replicates.
The hair regeneration success ratio was based on the vascularized grafts that were viable at the time of harvesting. The researcher who performed the quantification of success rates shown in Fig. RNA-seq data collection was performed by a separate core facility at Columbia Genome Center and analyzed without a prior knowledge of the conditions of the samples. No randomization was used. Data supporting the findings of this study are within this manuscript or available from the corresponding authors upon reasonable request.
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Microenvironmental reprogramming by three-dimensional culture enables dermal papilla cells to induce de novo human hair-follicle growth.
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