Force-Bioreactor for Assessing Pharmacological Therapies for Mechanobiological Targets.

Tissue fibrosis is a major health issue that impacts millions of people and is costly to treat. However, few effective anti-fibrotic treatments are available. Due to their central role in fibrotic tissue deposition, fibroblasts and myofibroblasts are the target of many therapeutic strategies centered primarily on either inducing apoptosis or blocking mechanical or biochemical stimulation that leads to excessive collagen production. Part of the development of these drugs for clinical use involves in vitro prescreening. 2D screens, however, are not ideal for discovering mechanobiologically significant compounds that impact functions like force generation and other cell activities related to tissue remodeling that are highly dependent on the conditions of the microenvironment. Thus, higher fidelity models are needed to better simulate in vivo conditions and relate drug activity to quantifiable functional outcomes. To provide guidance on effective drug dosing strategies for mechanoresponsive drugs, we describe a custom force-bioreactor that uses a fibroblast-seeded fibrin gels as a relatively simple mimic of the provisional matrix of a healing wound. As cells generate traction forces, the volume of the gel reduces, and a calibrated and embedded Nitinol wire deflects in proportion to the generated forces over the course of 6 days while overhead images of the gel are acquired hourly. This system is a useful in vitro tool for quantifying myofibroblast dose-dependent responses to candidate biomolecules, such as blebbistatin. Administration of 50 µM blebbistatin reliably reduced fibroblast force generation approximately 40% and lasted at least 40 h, which in turn resulted in qualitatively less collagen production as determined via fluorescent labeling of collagen.

Targeting Cell Contractile Forces: A Novel Minimally Invasive Treatment Strategy for Fibrosis.

Fibrosis is a complication of tendon injury where excessive scar tissue accumulates in and around the injured tissue, leading to painful and restricted joint motion. Unfortunately, fibrosis tends to recur after surgery, creating a need for alternative approaches to disrupt scar tissue. We posited a strategy founded on mechanobiological principles that collagen under tension generated by fibroblasts is resistant to degradation by collagenases. In this study, we tested the hypothesis that blebbistatin, a drug that inhibits cellular contractile forces, would increase the susceptibility of scar tissue to collagenase degradation. Decellularized tendon scaffolds (DTS) were treated with bacterial collagenase with or without external or cell-mediated internal tension. External tension producing strains of 2-4% significantly reduced collagen degradation compared with non-tensioned controls. Internal tension exerted by human fibroblasts seeded on DTS significantly reduced the area of the scaffolds compared to acellular controls and inhibited collagen degradation compared to free-floating DTS. Treatment of cell-seeded DTS with 50 mM blebbistatin restored susceptibility to collagenase degradation, which was significantly greater than in untreated controls (p < 0.01). These findings suggest that therapies combining collagenases with drugs that reduce cell force generation should be considered in cases of tendon fibrosis that do not respond to physiotherapy.

Substrate deformations induce directed keratinocyte migration.

Cell migration is an essential part of many (patho)physiological processes, including keratinocyte re-epithelialization of healing wounds. Physical forces and mechanical cues from the wound bed (in addition to biochemical signals) may also play an important role in the healing process. Previously, we explored this possibility and found that polyacrylamide (PA) gel stiffness affected human keratinocyte behaviour and that mechanical deformations in soft (approx. 1.2 kPa) PA gels produced by neighbouring cells appeared to influence the process of de novo epithelial sheet formation. To clearly demonstrate that keratinocytes do respond to such deformations, we conducted a series of experiments where we observed the response of single keratinocytes to a prescribed local substrate deformation that mimicked a neighbouring cell or evolving multicellular aggregate via a servo-controlled microneedle. We also examined the effect of adding either Y27632 or blebbistatin on cell response. Our results indicate that keratinocytes do sense and respond to mechanical signals comparable to those that originate from substrate deformations imposed by neighbouring cells, a finding that could have important implications for the process of keratinocyte re-epithelialization that takes place during wound healing. Furthermore, the Rho/ROCK pathway and the engagement of NM II are both essential to substrate deformation-directed keratinocyte migration.

Mouse Keratinocytes Without Keratin Intermediate Filaments Demonstrate Substrate Stiffness Dependent Behaviors.

INTRODUCTION: Traditionally thought to serve active vs. passive mechanical functions, respectively, a growing body of evidence suggests that actin microfilament and keratin intermediate filament (IF) networks, together with their associated cell-cell and cell-matrix anchoring junctions, may have a large degree of functional interdependence. Therefore, we hypothesized that the loss of keratin IFs in a knockout mouse keratinocyte model would affect the kinematics of colony formation, i.e., the spatiotemporal process by which individual cells join to form colonies and eventually a nascent epithelial sheet. METHODS: Time-lapse imaging and deformation tracking microscopy was used to observe colony formation for both wild type (WT) and keratin-deficient knockout (KO) mouse keratinocytes over 24 h. Cells were cultured under high calcium conditions on collagen-coated substrates with nominal stiffnesses of ~ 1.2 kPa (soft) and 24 kPa (stiff). Immunofluorescent staining of actin and selected adhesion proteins was also performed. RESULTS: The absence of keratin IFs markedly affected cell morphology, spread area, and cytoskeleton and adhesion protein organization on both soft and stiff substrates. Strikingly, an absence of keratin IFs also significantly reduced the ability of mouse keratinocytes to mechanically deform the soft substrate. Furthermore, KO cells formed colonies more efficiently on stiff vs. soft substrates, a behavior opposite to that observed for WT keratinocytes. CONCLUSIONS: Collectively, these data are strongly supportive of the idea that an interdependence between actin microfilaments and keratin IFs does exist, while further suggesting that keratin IFs may represent an important and under-recognized component of keratinocyte mechanosensation and the force generation apparatus.

Treatment with the cancer drugs decitabine and doxorubicin induces human skin keratinocytes to express Oct4 and the OCT4 regulator mir-145.

Previously, we showed that transient transfection with OCT4 not only produced high expression of Oct4 in skin keratinocytes, but also caused a generalized demethylation of keratinocyte DNA. We hypothesized that DNA demethylation alone might allow expression of endogenous OCT4. Here, we report that treatment with the cancer drug decitabine results in generalized DNA demethylation in skin keratinocytes, and by 48 h after treatment, 96% of keratinocytes show expression of the endogenous Oct4 protein and the OCT4 repressor mir-145. This is true for keratinocytes only, as skin fibroblasts treated similarly show no OCT4 or mir-145 expression. Decitabine-treated keratinocytes also show increased mir-302c and proliferation similar to other Oct4(+) cells. Treatment with doxorubicin, another cancer drug, induces expression of mir-145 only in cells that already express OCT4, suggesting that Oct4 regulates its own repressor. Co-treatment with decitabine and doxorubicin results first in increased OCT4 and mir-145, then a decrease in both, suggesting that OCT4 and mir-145 regulate each other. The novel strategy presented here provides a regulatable system to produce Oct4(+) cells for transformation studies and provides a unique method to study the effects of endogenous Oct4 in cancer cells and the surrounding somatic cells.

Discovery of SMAD4 promoters, transcription factor binding sites and deletions in juvenile polyposis patients.

Inactivation of SMAD4 has been linked to several cancers and germline mutations cause juvenile polyposis (JP). We set out to identify the promoter(s) of SMAD4, evaluate their activity in cell lines and define possible transcription factor binding sites (TFBS). 5'-rapid amplification of cDNA ends (5'-RACE) and computational analyses were used to identify candidate promoters and corresponding TFBS and the activity of each was assessed by luciferase vectors in different cell lines. TFBS were disrupted by site-directed mutagenesis (SDM) to evaluate the effect on promoter activity. Four promoters were identified, two of which had significant activity in several cell lines, while two others had minimal activity. In silico analysis revealed multiple potentially important TFBS for each promoter. One promoter was deleted in the germline of two JP patients and SDM of several sites led to significant reduction in promoter activity. No mutations were found by sequencing this promoter in 65 JP probands. The predicted TFBS profiles for each of the four promoters shared few transcription factors in common, but were conserved across several species. The elucidation of these promoters and identification of TFBS has important implications for future studies in sporadic tumors from multiple sites, and in JP patients.

Discovery of the BMPR1A promoter and germline mutations that cause juvenile polyposis.

Juvenile polyposis (JP) is an autosomal dominant hamartomatous polyposis syndrome where affected individuals are predisposed to colorectal and upper gastrointestinal cancer. Forty-five percent of JP patients have mutations or deletions involving the coding regions of SMAD4 and BMPR1A, but the genetic basis of other cases is unknown. We set out to identify the JP gene in a large kindred having 10 affected members without SMAD4 or BMPR1A coding sequence mutations or deletions. We found a germline deletion segregating in all affected members, mapping 119 kb upstream of the coding region of BMPR1A by multiplex ligation-dependent probe amplification and comparative genomic hybridization. To further understand the genomic structure of BMPR1A, we performed 5' RACE from lymphoblastoid cell lines and normal colon tissue, which revealed four non-coding (NC) exons and two putative promoters. Further analysis of this deletion showed that it encompassed 12 433 bp, including one promoter and NC exon. The activities of each promoter and deletion constructs were evaluated by luciferase assays, and the stronger promoter sequence analyzed for changes in JP patients without SMAD4 or BMPR1A alterations. A total of 6 of 65 JP probands were found to have mutations affecting this promoter. All probands examined had diminished BMPR1A protein by ELISA, and all promoter mutations but one led to significantly reduced luciferase activity relative to the wild-type promoter reporter. We conclude that we have identified the promoter for BMPR1A, in which mutations may be responsible for as many as 10% of JP cases with unknown mutations.

A family with two consecutive nonsense mutations in BMPR1A causing juvenile polyposis.

We describe a novel germline mutation of BMPR1A in a family with juvenile polyposis and colon cancer. This mutation consists of two consecutive substitutions (735-6 TG>AT) that cause two nonsense mutations (Y245X, G246X), inherited in an autosomal dominant fashion, on one parental chromosome. This mutation caused protein truncation, and represents a novel case of consecutive nonsense mutations in human disease.

HSP70 and EndoG modulate cell death by heat in human skin keratinocytes in vitro.

We examined how young and old keratinocytes died from heat stress in vitro. We found that keratinocyte cell death was not due to oxidative stress as neither Mn-SOD nor Cu-Zn-SOD was produced in either young or old heated keratinocytes. Instead, analysis of the anti-apoptotic factors, Bcl2 and HSP70, and the pro-apoptotic factors, caspase 3, caspase 8, Apaf-1, cytochrome c, AIF, and EndoG, indicated that keratinocyte cell death occurred via the caspase-independent EndoG apoptotic pathway. We found that both young and old keratinocytes died via the same pathway, and that we could specifically reduce both young and old keratinocyte death by addition of the EndoG inhibitor NEM. Further analysis suggested that the difference between young and old keratinocyte death was due to the synthesis of HSP70 protein, with the increase in response to heat more pronounced in young keratinocytes than in old keratinocytes. When we inhibited HSP70 by adding quercetin, death was increased in both young and old keratinocytes, but more so in old keratinocytes. These data suggest that old keratinocytes may die more readily than young keratinocytes when heated because they synthesize HSP70 at a lower efficiency. Such findings suggest that HSP70 production may be age-dependent.

Epidermal stem cells have the potential to assist in healing damaged tissues.

Homeostasis of continuously renewing tissues, such as the epidermis, is maintained by somatic undifferentiated, self-renewing stem cells, which are thought to persist throughout life. Through a series of labeling experiments, we previously showed that stem cells from mouse skin did not divide often, but they did divide at a steady rate in vivo. Using our recently redefined sorting method, we isolated epidermal stem and transit amplifying (TA) cells from mouse skin. When injected into a developing blastocyst or into damaged tissues, the stem cells, but not the TA cells, could participate in the formation of new tissues. We hypothesize that all tissues contain reserved undifferentiated stem cells that are primed to react if needed. These reserve stem cells could restore the tissue in which they reside or they could be called upon to help restore another tissue that was severely damage.

Human skin and gingival keratinocytes show differential regulation of matrix metalloproteinases when combined with fibroblasts in 3-dimensional cultures.

BACKGROUND: Matrix metalloproteinases (MMP) and their inhibitors are expressed in tissues during interactions between keratinocytes and fibroblasts. Maintaining the balance between MMPs and their inhibitors is critical; failure to do so can lead to severe tissue damage or complete destruction, as seen in periodontal disease. Previously we showed that 3-dimensional (3-D) cultures of homotypically-combined skin and gingival cells mimicked the tissues in protein and lipid production, but heterotypic cultures did not. METHODS: We examined the production and activation of MMPs in these homotypic and heterotypic combinations of skin and gingival keratinocytes and fibroblasts during the critical time that they reformed the tissues. Primary fibroblasts and keratinocytes were isolated from normal human gingiva and skin and grown in 3-D cultures for up to 42 days. MMP-1, MMP-2, and MMP-9 in the media and inhibition of MMPs from these cultures were analyzed. RESULTS: These experiments determined that skin fibroblasts grown with skin or gingival keratinocytes secrete increased amounts of MMP-1 compared to gingival fibroblasts; that the interaction of keratinocytes with fibroblasts decreases the amount of MMP-2 produced by the fibroblasts in 3-D cultures; that skin keratinocytes, but not gingival keratinocytes, interact with fibroblasts to upregulate expression of the active form of MMP-9; and that medium conditioned by gingival 3-D cultures does not contain an inhibitor of MMP-9. CONCLUSION: Varying the type of fibroblast beneath the keratinocytes allowed us to determine that skin and gingival keratinocytes differentially regulate the production and activation of MMP-9, but not MMP-2, a finding that could influence the success of tissue grafting after periodontal surgery.

As epidermal stem cells age they do not substantially change their characteristics.

In this study, we ask the basic question: do stem cells age? We demonstrated that epidermal stem cells isolated from neonatal mice had the capacity to form multiple cell lineages during development. Here we demonstrate the cell lineages are clonal, and that epidermal stem cells isolated from the footpad epithelium of old mice have similar capabilities. Using Hoechst dye exclusion and cell size, we isolated viable homogenous populations of epidermal stem and transit-amplifying (TA) cells from GFP-transgenic mice, and injected these cells into 3.5-d blastocysts. Only the stem-injected blastocysts produced mice with GFP+ cells in their tissues. Furthermore, aged and young stem cells showed similar gene and protein expression profiles that showed some differences from the TA cell profiles. These data suggest that there may be a fundamental difference between somatic stem and TA cells, and that an epidermal stem cell placed in a developmental environment can alter its fate determination no matter what its age.

Mouse epidermal stem cells proceed through the cell cycle.

The epidermis is a continuously renewing tissue maintained by undifferentiated stem cells. For decades it has been assumed that epidermal stem cells (ESCs) were held in the G0 phase of the cell cycle and that they only entered the cell cycle when needed. Previously, we showed that ESCs retained nuclear label for long periods, indicating that these cells did not proceed through the cell cycle at the same rate as the other proliferative basal cells. However, their exact cell-cycle profile has not been determined because a pure population of ESCs has not been available. In this study, we sorted stem and transient amplifying (TA) cells from murine neonatal back skin, and adult ear, footpad, and back skin, using our recently developed method. We found that neonatal back skin had two times the number of ESCs as the adult tissues. Despite the age and anatomical difference, these ESC populations exhibited similar cell cycle profiles with approximately 96% in G0/G1 and 4% in S-G2/M. The cell cycle profiles of the TA cells from neonatal back skin and adult footpad also showed a profile similar to each other (85% in G1 and 15% in S-G2/M). Examination of genes on a cell cycle chip showed that proliferation associated genes and only p57 were upregulated in the TA cell and ESC population, respectively. We found BrdU positive and cyclin B1 positive cells in all groups, confirming that both ESCs and TA cells were cycling. These data demonstrate that there are more TA cells dividing than ESCs, that the cell cycle profile of adult TA cells is related to the proliferative state of the tissue in which they reside, and that ESC proceed through the cell cycle.

Recapitulation of oral mucosal tissues in long-term organotypic culture.

To test the influence of fibroblasts on epithelial morphology and expression of keratinocyte proteins and barrier lipids, we bioengineered homotypic and heterotypic oral mucosae and skin using cultured adult human cells. Fibroblasts were allowed to modify collagen type I gels for 2 weeks before keratinocytes were added. The organotypic cultures were then grown at the air-liquid interface for 4 weeks. In homotypic combinations, epithelial morphology and protein expression closely mimicked those in vivo. In heterotypic combinations, the morphology resembled that in vivo and keratinocytes expressed their typical markers, except when skin keratinocytes were recombined with alveolar fibroblasts; they expressed K19, K4, and K13, which is similar to oral mucosal epithelia rather than to the epidermis. Morphologically, the stratum corneum layers were typical for the epithelial tissues. Grafting the bioengineered cultures to the backs of Nude mice did not change the results, suggesting that our findings are not merely a culture phenomenon. Lipid profiles of the homotypic combinations mimicked the profiles found in the normal epithelial tissues, except that the engineered alveolar epithelium expressed more ceramide 2 than that in vivo. In the heterotypic combinations, keratinocytes appeared to control the lipid profile, except in the combination of skin keratinocytes with alveolar fibroblasts, wherein the ceramide profile appeared to be partly that of alveolar epithelium and partly that of epidermis. These results suggest that cultured adult fibroblasts and keratinocytes are sufficient to recapitulate graftable oral tissues, and, except for alveolar fibroblasts, the type of fibroblast had little influence on keratinocyte differentiation.