Establishment of an in vitro organoid model of dermal papilla of human hair follicle.

Human hair dermal papilla (DP) cells are specialized mesenchymal cells that play a pivotal role in hair regeneration and hair cycle activation. The current study aimed to first develop three-dimensional (3D) DP spheroids (DPS) with or without a silk-gelatin (SG) microenvironment, which showed enhanced DP-specific gene expression, resulting in enhanced extracellular matrix (ECM) production compared with a monolayer culture. We tested the feasibility of using this DPS model for drug screening by using minoxidil, which is a standard drug for androgenic alopecia. Minoxidil-treated DPS showed enhanced expression of growth factors and ECM proteins. Further, an attempt has been made to establish an in vitro 3D organoid model consisting of DPS encapsulated by SG hydrogel and hair follicle (HF) keratinocytes and stem cells. This HF organoid model showed the importance of structural features, cell-cell interaction, and hypoxia akin to in vivo HF. The study helped to elucidate the molecular mechanisms to stimulate cell proliferation, cell viability, and elevated expression of HF markers as well as epithelial-mesenchymal crosstalks, demonstrating high relevance to human HF biology. This simple in vitro DP organoid model system has the potential to provide significant insights into the underlying mechanisms of HF morphogenesis, distinct molecular signals relevant to different stages of the hair cycle, and hence can be used for controlled evaluation of the efficacy of new drug molecules.

A non-invasive method to evaluate the efficacy of human myoblast in botulinum-A toxin induced stress urinary incontinence model in rats.

PURPOSE: To develop a simple non-invasive method to assess the efficacy of a cell based therapy for treating stress urinary incontinence (SUI). MATERIALS AND METHODS: In this study, skeletal myoblasts were used as candidate therapy to reverse SUI. The SUI model was created in rats using periurethral injection of botulinum-A toxin injection. Two weeks later, the rats were administered saline and the level of continence in each botulinum-A toxin treated and control animals was assessed by the extent of voiding using metabolic cages. To determine the efficacy of myoblasts to reverse SUI, botulinum-A toxin treated incontinent rats were injected with either cultured human skeletal myoblasts or with buffered saline (sham control). Two weeks post implantation, the extent of continence was evaluated as mentioned above. RESULTS: The difference in void volume between botulinum-A toxin -treated and control rats were significant. Histological analysis of the urethra showed remarkable atrophy of the muscular layer. A significant reversal (P = .025) in the volume of voiding was observed in cell-implanted rats as compared to sham injected rats. Histological analysis of the urethra implanted with myoblasts showed recovery of the atrophied muscular layer in comparison to sham control. Immunofluorescence analysis of the cell injected tissues confirmed the presence of human myoblasts in the regenerated area. CONCLUSION: This simplified method of in vivo testing can serve as a tool to test the efficacy of new therapies for treating SUI.

In vitro and in vivo evaluation of (L)-lactide/ε-caprolactone copolymer scaffold to support myoblast growth and differentiation.

Skeletal muscle regeneration involves the activation of satellite cells to myoblasts, followed by their proliferation and fusion to form multinucleated myotubes and myofibers. The potential of in vitro proliferated myoblasts to treat various diseases and tissue defects can be exploited using tissue-engineering principles. With an aim to develop a biocompatible and biodegradable scaffold that supports myoblast growth and differentiation, we have developed a porous sponge with 70/30 L-lactide/ε-caprolactone copolymer (PLC) using a phase inversion combined with particulate leaching method. Degradation studies indicated that the sponge retained its structural integrity for 5 months in vitro and had undergone complete biodegradation within 9 months in vivo. The sponge supported human myoblasts attachment and its proliferation. Myoblasts seeded on the PLC sponge differentiated and fused in vitro to form myotubes expressing myosin heavy chain. Histological and molecular analyses of the PLC scaffolds seeded with green fluorescent protein-labeled human myoblasts and implanted ectopically under the skin in SCID mice demonstrated the presence of multinucleated myotubes expressing human muscle-specific markers. Our results suggest that PLC sponges loaded with myoblasts can be used for skeletal muscle engineering or for inducing muscle repair.

The anti-motility signaling mechanism of TGFß3 that controls cell traffic during skin wound healing.

When skin is wounded, migration of epidermal keratinocytes at the wound edge initiates within hours, whereas migration of dermal fibroblasts toward the wounded area remains undetectable until several days later. This "cell type traffic" regulation ensures proper healing of the wound, as disruptions of the regulation could either cause delay of wound healing or result in hypertrophic scars. TGFß3 is the critical traffic controller that selectively halts migration of the dermal, but not epidermal, cells to ensure completion of wound re-epithelialization prior to wound remodeling. However, the mechanism of TGFß3's anti-motility signaling has never been investigated. We report here that activated TßRII transmits the anti-motility signal of TGFß3 in full to TßRI, since expression of the constitutively activated TßRI-TD mutant was sufficient to replace TGFß3 to block PDGF-bb-induced dermal fibroblast migration. Second, the three components of R-Smad complex are all required. Individual downregulation of Smad2, Smad3 or Smad4 prevented TGFß3 from inhibiting dermal fibroblast migration. Third, Protein Kinase Array allowed us to identify the protein kinase A (PKA) as a specific downstream effector of R-Smads in dermal fibroblasts. Activation of PKA alone blocked PDGF-bb-induced dermal fibroblast migration, just like TGFß3. Downregulation of PKA's catalytic subunit nullified the anti-motility signaling of TGFß3. This is the first report on anti-motility signaling mechanism by TGFß family cytokines. Significance of this finding is not only limited to wound healing but also to other human disorders, such as heart attack and cancer, where the diseased cells have often managed to avoid the anti-motility effect of TGFß.

TbetaRI/Alk5-independent TbetaRII signaling to ERK1/2 in human skin cells according to distinct levels of TbetaRII expression.

TGFß binding to the TGFß receptor (TßR) activates R-Smad-dependent pathways, such as Smad2/3, and R-Smad-independent pathways, such as ERK1/2. The mechanism of the TGFß-TßRII-TßRI-Smad2/3 pathway is established; however, it is not known how TGFß activates ERK1/2. We show here that although TGFß equally activated Smad2/3 in all cells, it selectively activated ERK1/2 in dermal cells and inhibited ERK1/2 in epidermal cells. These opposite effects correlated with the distinct expression levels of TßRII, which are 7- to 18-fold higher in dermal cells than in epidermal cells. Reduction of TßRII expression in dermal cells abolished TGFß-stimulated ERK1/2 activation. Upregulation of TßRII expression in epidermal cells to a similar level as that in dermal cells switched TGFß-induced ERK1/2 inhibition to ERK1/2 activation. More intriguingly, in contrast to the equal importance of TßRII in mediating TGFß signaling to both Smad2/3 and ERK1/2, knockdown of TßRI/Alk5 blocked activation of only Smad2/3, not ERK1/2, in dermal cells. Similarly, expression of the constitutively activated TßRI-TD kinase activated only Smad2/3 and not ERK1/2 in epidermal cells. This study provides an explanation for why TGFß selectively activates ERK1/2 in certain cell types and direct evidence for TßRI-independent TßRII signaling to a R-Smad-independent pathway.

Transforming growth factor alpha (TGFalpha)-stimulated secretion of HSP90alpha: using the receptor LRP-1/CD91 to promote human skin cell migration against a TGFbeta-rich environment during wound healing.

Jump-starting and subsequently maintaining epidermal and dermal cell migration are essential processes for skin wound healing. These events are often disrupted in nonhealing wounds, causing patient morbidity and even fatality. Currently available treatments are unsatisfactory. To identify novel wound-healing targets, we investigated secreted molecules from transforming growth factor alpha (TGFalpha)-stimulated human keratinoytes, which contained strong motogenic, but not mitogenic, activity. Protein purification allowed us to identify the heat shock protein 90alpha (hsp90alpha) as the factor fully responsible for the motogenic activity in keratinocyte secretion. TGFalpha causes rapid membrane translocation and subsequent secretion of hsp90alpha via the unconventional exosome pathway in the cells. Secreted hsp90alpha promotes both epidermal and dermal cell migration through the surface receptor LRP-1 (LDL receptor-related protein 1)/CD91. The promotility activity resides in the middle domain plus the charged sequence of hsp90alpha but is independent of the ATPase activity. Neutralizing the extracellular function of hsp90alpha blocks TGFalpha-induced keratinicyte migration. Most intriguingly, unlike the effects of canonical growth factors, the hsp90alpha signaling overrides the inhibition of TGFbeta, an abundant inhibitor of dermal cell migration in skin wounds. This finding provides a long-sought answer to the question of how dermal cells migrate into the wound environment to build new connective tissues and blood vessels. Thus, secreted hsp90alpha is potentially a new agent for wound healing.

A "traffic control" role for TGFbeta3: orchestrating dermal and epidermal cell motility during wound healing.

Cell migration is a rate-limiting event in skin wound healing. In unwounded skin, cells are nourished by plasma. When skin is wounded, resident cells encounter serum for the first time. As the wound heals, the cells experience a transition of serum back to plasma. In this study, we report that human serum selectively promotes epidermal cell migration and halts dermal cell migration. In contrast, human plasma promotes dermal but not epidermal cell migration. The on-and-off switch is operated by transforming growth factor (TGF) beta3 levels, which are undetectable in plasma and high in serum, and by TGFbeta receptor (TbetaR) type II levels, which are low in epidermal cells and high in dermal cells. Depletion of TGFbeta3 from serum converts serum to a plasmalike reagent. The addition of TGFbeta3 to plasma converts it to a serumlike reagent. Down-regulation of TbetaRII in dermal cells or up-regulation of TbetaRII in epidermal cells reverses their migratory responses to serum and plasma, respectively. Therefore, the naturally occurring plasma-->serum-->plasma transition during wound healing orchestrates the orderly migration of dermal and epidermal cells.

Signals that initiate, augment, and provide directionality for human keratinocyte motility.

Human keratinocytes (HK) migration plays a critical role in the re-epithelialization of acute skin wounds. Although extracellular matrices (ECM) and growth factors (GF) are the two major pro-motility signals, their functional relationship remains unclear. We investigated how ECM and GF regulate HK motility under defined conditions: (1) in the absence of GF and ECM and (2) with or without GF with cells apposed to a known pro-motility ECM. Our results show that HK migrate on selected ECM even in the total absence of GF. This suggests that certain ECM alone are able to "initiate" HK migration. Unlike ECM, however, GF alone cannot initiate HK migration. HK cannot properly migrate when plated in the presence of GF, regardless of the concentration, without an ECM substratum. The role of GF, instead, is to augment ECM-initiated motility and provide directionality. To gain insights into the mechanism of action by ECM and GF, we compared, side-by-side, the roles of three major mitogen-activated protein kinase cascades, extracellular-signal-regulated kinase (ERK)1/2, p38, and c-Jun N-terminal kinase (JNK). Our data show that ERK1/2 is involved in mediating collagen's initiation signal and GF's augmentation signal. p38 is specific for GF's augmentation signal. JNK is uninvolved in HK motility. Constitutively activated p38 and ERK1/2 alone could not initiate HK migration. Co-expression of both constitutively activated p38 and ERK1/2, however, could partially mimic the pro-motility effects of collagen and GF. This study reveals for the first time the specific functions of ECM and GF in cell motility.