WO2010081152A2 - Keloid derived precursor cells and its applications of use thereof - Google Patents

Abstract

The present invention relates to keloid derived precursor cells (KPCs), which exhibit clonogenicity, self-renewal, distinct embryonic and mesenchymal stem cell surface markers, and multipotent differentiation. More specifically, the invention provides methods of utilizing KPCs for the generation of a human keloid-like tumor model, which allows for testing of the anti-tumor therapeutic effect of antibodies targeting distinct niche components, such as IL-6 and IL-17.

Description

KELOID DERIVED PRECURSOR CELLS AND ITS APPLICATIONS

OF USE THEREOF

The present application claims the benefit of the filing date of U.S.

Provisional Application No. 61/144,091 filed January 12, 2009 and U.S. Provisional Application No. 61/240, 141, filed September 4, 2009, the disclosure of which are incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under Contract No. SIl AR47359 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD OF THE INVENTION The invention relates in general to keloid derived precursor cells

(KPC). More specifically, the invention provides compositions comprising KPC and methods of using KPC for ή^'broproliferative diseases. BACKGROUND OF THE INVENTION

The fate of the stem cells, undergoing self- renewal or differentiation, is dependent on a specialized microenvironment or niche in which the cells reside [I]. The stem cell niche [2] encompasses all elements immediately surrounding the stem cells, including non-stem cells, the extracellular matrix (ECM), as well as soluble molecules present in the locale [3, 4]. Under homeostatic or physiological conditions, the extrinsic niche components or growth factors protect the stem cells from excessive proliferation by providing a balanced proliferation- inhibiting and proliferation-promoting signal [5, 6]. Meanwhile, the stem cells must periodically activate to produce specific lineage progenies for the regeneration or repair of tissues [3]. Therefore, the maintenance of a steady state between stem cell quiescence and activity is the hallmark of a functionally normal niche [6], whereas the deregulation of niche signals may lead to the uncontrolled self-renewal and proliferation of the stem cells, thus contributing to the emergence of the so-called niche-generated diseases including pre-cancer and tumorigenesis [4, 7].

Interleukin (IL)-6 is produced at the site of acute inflammation [8] and plays a critical role in both cellular and humoral immune responses [9]. IL- 6 can trigger the switch from acute to chronic inflammation by enhancing the recruitment of monocytes [10], therefore, serves as a key player in both acute and chronic inflammation [H]. Several studies have reported elevated IL-6 levels in several inflammatory diseases, including chronic inflammatory and fibro -proliferative diseases such as keloids [12, 13], rheumatoid arthritis, inflammatory bowel disease (colitis), multiple sclerosis, and pulmonary fibrosis [14, 15]. Aside from its established function as an immuno- modulator, IL-6 may contribute to the regulation of stem cell functions via several pathways leading to the inhibition of lymphocyte apoptosis [16] and the maintenance of hematopoietic stem cells [17]. Most recently, IL-6 has been reported as a critical tumor promoter during early colitis-associated tumorigenesis [18, 19]. Immune cells, which often infiltrate tumor and pre-neoplastic lesions, are capable to perpetuate a localized inflammatory response via a variety of cytokines and chemokines, and enhance the growth and survival of pre malignant cells. IL- 6 has also been demonstrated to trigger malignant features in Notch-3- expressing stem/progenitor cells from human ductal breast carcinoma and normal mammary gland [20] and further up-regulates telomerase activity in human malignancies [21, 22]. Since telomerase plays an essential role in the regulation of cell proliferation and senescence [23, 24] it has been routinely used as a functional marker to assess stem cell function and tissue homeostasis [25, 26].

IL- 17, a recently discovered pro-inflammatory cytokine, is secreted by a distinct subtype of activated CD4+ T-cells known as Thl7 [27, 28], Most recently, several studies have clearly demonstrated the critical role of TGF- β and IL-6 or other inflammatory cytokines in the differentiation of human Thl7 cells [29-32]. The finding of this cytokine has changed our perspectives on chronic inflammatory diseases, including inflammatory bowel disease, rheumatoid arthritis, psoriasis, multiple sclerosis, and allergic skin immune responses [33-36], IL- 17 is produced exclusively by activated T cells, however, its receptor is ubiquitously expressed in many cell types, thus making them potential targets [30, 37]. IL-17 amplifies the immune response mediated by a variety of cytokines such as IL-6, tumor necrosis factor (TNF)-α and IL- 16, chemokines such as monocyte chemoattachment protein- 1 (MCP-I) and macrophage inflammatory protein-2 (MIP-2)/IL-8, cell-surface markers such as intercellular adhesion molecule-1 (ICAM-I), and pro- inflammatory mediators including prostaglandin E2, nitric oxide, cyclooxygenase-2, and C-reactive protein [38- 42], Among these inflammatory cytokines, IL-6 seems to be a major IL-17 signaling target in a variety of cells including macrophages, fibroblasts, osteoblasts, epithelial cells, and chondrocytes [39, 40, 43-46]. More importantly, IL-6 not only functions upstream of IL-17 but also acts as a critical downstream target of IL-17, thus forming a paracrine/autocrine feedback loop that promotes autoimmune and allergic diseases [45, 46], In addition to its immuno-regulatory role, IL-17A not only promotes the proliferation of human bone marrow mesenchymal stem cells (BMMSCs) but also induces their migration, motility, and osteodifferentiation [47]. Based on existing findings we asked whether IL-17 and IL-6 coordinately interact and contribute to the persistent local tissue inflammation in keloid, i.e. sustaining the "pathological" niche, therefore support the proliferation- promoting signal of the benign dermal growth.

Keloid is an exuberant scar unique to human with highest incidence in African-Americans, resembles benign tumor behavior by its aggressive dermal growth that continues to expand beyond the boundaries of the original wound margins (Figure 8A), rarely regresses as observed in hypertrophic scars [48], and recurs at a high rate of up to 80% after surgical removal [49]. These dermal growths, similar to other fibroid tumors, could potentially reach a grotesque size in the craniofacial region with tremendous esthetic, functional, and psychologically debilitating sequelae [48]. As a chronic inflammatory and fibro-proliferative disease (Figure 8A), keloids exhibit distinctive histological features characterized by a high density of mesenchymal cells, an abundant ECM stroma, a local infiltration of inflammatory cells including mast cells and lymphocytes, and a milieu of enriched cytokines, especially transforming growth factor-61 (TGF-Bl) and IL-6 [12, 13]. Clinically, keloids have been correlated with some degree of inflammatory response associated with tissue injury and most often, manifested clinically as a tender, painful, pruritic or burning sensation. We believe that the altered or "pathological niche" driven by the inflammatory cytokine IL-17/IL-6 axis potentially regulates the benign, yet aggressive growth behavior of the dermal tumor. In addition, the fibroproliferative nature of keloids suggests that mesenchymal stem cells may serve as primary source of multipotent cells to rapidly repopulate the wound site in response to exogeneous insults, such as trauma, surgical injury, or infection. Therefore, keloids provide an ideal benign tumor model to study the role of stem cells and their special niche components in promoting and maintaining the proliferative growth of the dermal scar. SUMMARY OF THE INVENTION

In one embodiment, the invention relates to keloid derived precursor cells (KPC) with stem cell properties comprising clonogenicity, multiple differentiation capacity, and self-renewal. KPC are capable of multiple differentiation into mesoderm-derived adipocytes, osteoblasts, smooth muscle cell-like cells, and ectoderm-derived different types of neural cells.

In another embodiment, the invention relates to a keloid animal model that may be used for the treatment and prevention of keloid scarring. The method of treating keloid scarring comprises obtaining a keloid animal model, administering an IL-6 neutralizing antibody into the keloid scar of the model, and determining that a reduction in the size of the scar is indicative that the scar has been treated.

The method of preventing keloid scarring comprises administering an IL-6 neutralizing antibody into an immunocompromised mouse, transplanting isolated KPC into the mouse and allowing them to grow and form a keloid scar, administering an IL-6 neutralizing antibody into the scar of the mouse, comparing the growth of the keloid scar in the mouse given the IL-6 neutralizing antibody with the growth of a keloid in a control mouse, and determining that the amount of keloid scarring in the mouse given the IL-6 neutralizing antibody is less than the amount of keloid scarring in the control mouse is indicative of the prevention of keloid scarring.

In a related embodiment, the invention relates to methods of treating keloid scarring, comprising decreasing IL-6 levels in a keloid scar by administering an IL-6 neutralizing antibody into the scar, and determining that a reduction in the size of the scar is indicative that the scar has been treated.

In accordance with another embodiment, the invention relates to methods of preventing keloid scarring from occurring. The method comprises administering IL-6 neutralizing antibody into a subject, administering KPC into the subject, comparing the growth of KPC in the subject given the IL-6 neutralizing antibody with the growth of KPC in a control subject, and determining that the amount of keloid scarring in the subject given the IL-6 neutralizing antibody is less than the amount of keloid scarring in the control subject is indicative of the prevention of keloid scarring.

Within one aspect, the present invention provides methods of screening for a pharmacological target of inflammatory diseases. The method comprises contacting a pharmacological target with a IL-17/IL-6 inflammatory niche model and determining that changes in the amount of inflammation or an agent that causes inflammation, is indicative of the identification of a pharmacological target that may be used to treat an inflammatory disease.

The above-mentioned and other features of this invention and the manner of obtaining and using them will become more apparent, and will be best understood, by reference to the following description, taken in conjunction with the accompanying drawings. The drawings depict only typical embodiments of the invention and do not therefore limit its scope. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Identification of dermal derived precursor cells from keloid tissues. A, Frozen sections of keloid tissues and their matched peripheral normal skins were immunostained with specific antibodies for human Oct-4 and SSEA-4. Scale bars, 50μm. B, Expression of Oct-4 and SSEA-4 in keloids (Kl₇ K2) and matched normal skins (Nl, N2) were determined by Western blot analysis and scanning densitometer. C, Colony formation analysis of cells derived from keloids and normal skin (mean ± SEM). * P < 0.05; ** P < 0.01. D, Cell population doubling numbers were determined in single cell colony- derived stem cells from keloids (KPCs) and matched peripheral normal skins (SKPs) by standard 3T3 cell culture protocol (mean ± SEM). E, Flow cytometric analysis of cell surface markers of KPCs and SKPs. F, RT-PCR (upper panel) and qPCR (lower panel) analysis of stem cell genes of KPCs (Kl, K2) and SKPs (Nl, N2). G and H, Analysis of telomerase activity using TeIoTAGGG telonierase PCR ELISA kit (mean ± SEM). * P < 0.05; ** P < 0.01; *** P < 0.001. Data are representative of at least five independent experiments using specimens obtained from different patient donors with matched normal controls (n=5).

Figure 2. In vivo transplantation of dermal stem cells. A, Size and volume of transplants generated from SKPs and KPCs using Gelfoam as carrier for 8 weeks (mean ± SEM), ** P < 0.01. B, Immunohistological studies of KPC transplant tissues using a specific antibody for human mitochondria (purple color) and type I collagen (brown color), respectively. Scale bars, 50μm. C, H & E histological stain of transplants. Scale bars, 50μm. D, In vivo bone regeneration by SKPs or KPCs using hydroxyapatite/tricalcium phosphate (HA/TCP) as carrier. Scale bars, 50μm. E, Serial transplantation of KPCs. KPCs (2 x 10⁶) with hydrogel were injected subcutaneously into nude mice. After 4 weeks, the transplanted were harvested and the recovered cells were expanded in vitro and re- transplanted into mice for another 4 weeks. The transplant tissues were harvested for H & E staining or immunohistochemical (IHC) staining with a specific antibody for human mitochondria (purple color). Scale bars, 50μm. The results are representative of five independent experiments.

Figure 3. Increased expression of IL-6 and IL-17 in keloids. A, Cytokine expression profiling of keloid and matched normal skin tissue lysates by human cytokine antibody array. B, Determination of IL-17 levels in keloid and normal skin tissue lysates by ELISA (mean ± SEM). * P<0.05. C, Immunohistochemical studies of IL-6 and IL-17 expression in keloid and matched normal skin tissues. Scale bars, 50μrα. D, Confocal immunofluorescence studies show co-localization of IL-17 around CD3⁺ cells as determined by dual-color staining. Scale bars, lOOμm. E and F, Western analysis of IL-17 (E) and IL-6 expression (F) in keloid tissues and matched normal skins. G and H, Expression of IL-6 and IL-6 receptor (IL-6R) in precursor cells derived from different keloid samples (Kl, K2) and matched normal skins (Nl, N2) as determined by Western blot (G) and RT-PCR analyses (H). I, Expression of IL-17 receptor (IL-17R) in precursor cells derived from different keloid samples (Kl, K2) and matched normal skins (Nl, N2) as determined by western blot and RT-PCR analyses. Data are representative of at least five independent experiments using KPCs and matched SKPs from different patient donors (n=5). Figure 4. IL-6 increases Oct-4 and telomerase expression in KPCs and SKPs. A, KPCs and SKPs were cultured in 1% FBS for 24 hours followed by exposure to different concentrations of IL-6, and BrdU incorporation in KPCs and SKPs was determined (mean ± SEM). ** P<0.01. B, Expression of Oct-4 mRNA and protein were determined by Western blotting (WB) and RT-PCR, respectively. C, Immunofluorescence studies of Oct-4 expression in SKP and KPC after incubated with 20ng/ml IL-6 for 24 hours. Scale bars, 20μm. D, Expression of hTERT mRNA and protein were determined by Western blotting (WB) and RT-PCR, respectively. E, Immunofluorescence studies of hTERT expression in SKP and KPC following incubation with 20ng/ml IL-6 for 24 hours. Scale bars, 20μm. F, Telomerase enzyme activity of SKPs and KPCs in response to IL-6 as determined by TeIoTAGGG Telomerase PCR ELISA. G and H, Treatment with neutralizing antibody for IL-6 (IL-6Ab) decreased the basal level of hTERT as determined by Western blotting (G) and TeIoTAGGG Telomerase PCR ELISA (H). An isotype-matched normal mice IgG (mlgG) was used as negative control (mean ± SEM). * P < 0.05; ** P < 0.01; *** P < 0.001; ns, no significance. The results are representative of at least five independent experiments using KTCs and the matched SKPs from different patient donors (n=5).

Figure 5. IL-17-induced hTERT and Oct-4 expression is IL-6- dependent. A, IL- 17 stimulated IL-6 secretion in SKPs and KPCs as determined by ELISA analysis (mean ± SEM). ** P < 0.01; ns, no significance. B₁ IL- 17 stimulated the expression of hTERT and Oct-4 in SKPs and KPCs as determined by Western blot analysis. C, Blocking IL-6, but not IL-17 attenuated the basal expression of hTERT and Oct-4 in SKPs and KPCs. Cells were treated with 5μg/mL of neutralizing antibody for either IL-6 (IL-6 Ab), or IL-17 (IL- 17Ab), or both for 24 hours and expression of hTERT and Oct-4 was determined by Western blot analysis. D, IL-17-induced upregulation of hTERT and Oct-4 expression in SKPs and KPCs was significantly attenuated by treatment with neutralizing antibodies for either IL-17, or IL-6, or both. After serum-starved for 24 hours, cells were pretreated with neutralizing antibodies, followed by incubation with 20ng/mL of IL-17. The expression of hTERT and Oct-4 was determined by Western blot analysis. E, IL- 6 -induced upregulation of hTERT and Oct-4 expression in SKPs and KPCs was attenuated only by IL- 6Ab, not by IL- 17Ab. Following serum -starvation for 24 hours, cells were pretreated with neutralizing antibodies and incubated with 20ng/mL of IL- 6. Expression of hTERT and Oct-4 was determined by Western blot analysis. An isotype-matched normal mouse IgG (mlgG) was used as negative controls. The results are representative of at least five independent experiments using KPCs and the matched SKPs from different patient donors (n=5).

Figure 6. In vivo effect of IL-6-linked hydrogel on growth of transplanted KPCs and SKPs. A, KPCs or SKPs (2 x 10⁶) were mixed with hydrogel incorporated with or without IL-6 and subcutaneously injected into immunocompromised mice for 8 weeks. Size and volume of transplants generated from KPCs and SKPs were measured (mean ± SEM). Scale bars, lmm. ** P < 0.01; *** P < 0.001. B, Histological analysis of KPC and SKP transplants. Scale bars, 50μm. C, Immunohistochemical studies (IHC) of transplants of KPCs and SKPs using antibody specific for human mitochondria (purple color) and type I collagen (brown color) or an isotype- matched control IgG (IgG isotype). Scale bars, 50μm. D, Increased synthesis and secretion of collagen by IL-6 in KPCs versus SKPs generated transplants as demonstrated by electron microscopy (EM). Scale bars, 500 nm. E, Expression of Oct-4 and hTERT in KPC and SKP transplants with or without IL-6 as determined by Western blot analysis. F, Expression of Oct-4 and hTERT in KPC and SKP transplants with or without IL-6 as determined by immunohistochemical studies. Scale bars, 50μm. G, Telomerase enzyme activity of SKPs and KPCs transplants with or without IL-6 as determined by TeIoTAGGG Telomerase PCR ELISA (mean ± SEM). * P < 0.05; ** P < 0.01. H, Increased expression of proliferating cell nuclear antigen (PCNA) in transplants of SKPs and KPCs in the presence of IL-6 as determined by immunohistochemical studies. Scale bars, 50μm. I and J, Treatment with neutralizing antibodies against IL-6 inhibited in vivo formation and growth maintenance of KPC transplants. KPCs were mixed with hydrogel with or without IL-6 neutralizing antibody (IL-6Ab, lOμg/ml) and injected subcutaneously into immunocompromised mice. KPC transplants were evaluated after 8 weeks (I). To test the suppressive effect of IL-6 neutralizing antibody on growth of KPC-derived tissues, IL-6 neutralizing antibody was locally injected into KPC-transplants twice a week (5μg/time) for another 4 weeks (J). An isotype-matched normal mice IgG (IgG isotype) was used as negative controls. The results are representative of five independent experiments using KPCs and the matched SKPs from different patient donors (mean ± SEM). ** PO.01; ns, no significance. Figure 7. Effect of T cell infusion on in vivo growth of KPC- generated transplants. A, Size and volume of KPC transplants (mean ± SEM). Activated CD4⁺CD25- T lymphocytes were infused at 1 x 10⁶/mouse through the tail vein two days before KPC transplants. KPCs were mixed with hydrogel with or without IL-6 and transplanted subcutaneously for 8 weeks. * P < 0.05; ** P < 0.01. B, Dual-color immunostaining for mouse CD4 (red) and IL- 17 (green) using paraffin-embedded sections of KPC transplants. Scale bars, 50μm. (C) Tissue lysates were prepared from the transplant tissues, and IL- 17 expression was analyzed by ELISA (mean ± SEM). * P<0.05, ** P<0.01. The results are representative of five independent experiments using KPCs and the matched SKPs from different patient donors.

Figure 8. Isolation of precursor cells from keloid tissues. A, Keloidscar displays benign tumor phenotype in terms of growth and recurrence. B, H&E histological stain of keloidtissues and the matched peripheral normal skins. PD: papillary dermis; RD: reticular dermis. Scale bars, 50μm. C, Semi-quantification of immunohistochemicalstaining of Oct- 4 and SSEA-4 in keloidand the matched normal skin {Fig. IA) as described in Materials &Methods. **JP<0.01. D, Colony formation of stem cells derived from keloid(KPCs) and normal skin (SKPs). E, Subcloning and culture of mesenchymalstem cells from keloids(KPCs) in α-MEM medium supplemented with 10% FBS, 1 x NEAA (non-essential amino acid) and antibiotics. Scale bars, lOOμm. F, Determination of doubling time of KPCsand SKPs as described in Materials and Methods (mean ±SEM). G, Karyotypingof the SKP and KPC clones at passage 10. The results are representative of at least five independent experiments.

Figure 9. Multipotent differentiation of keloidderived precursor cells (KPCs). _4-C,Adipogenicdifferentiation of SKP or KPCas determined by Oil Red O staining (A and B) and RT-PCR analysis of specific adipocytegenes (C). -D-i^Osteogenicdifferentiation as determined by Alizarin Red S staining (D andE) and RT-PCR analysis of osteocalcingene (F). Human bone marrow mesenchymalstem cells (hBMMSCs) were used as positive controls whereas KPCsculturing under normal growth medium were served as non-induction control.Scale bars, 50μm. Data are representative of at least five independent experiments using KPCsand the matched SKPs from 5 different patient donors (mean ±SEM). * P< 0.05; ns, no significance.

Figure 10. Sphere-colony formation of KPCs. A, Subcloningand expansion of sphere-colonies derived from keloidsin DMEM-LG/F12 (3:1) supplemented with 40ng/mLFGF-2, 20ng/mLEGF_; B27 and antibiotics. Scale bars, lOOμm. .Band C, Expression of stem cell markers and BrdUincorporation by keloid-derived sphere colonies (K1-K3) as determined by immunofluorescencestaining (B) and RT-PCR analysis (C). Scale bars, 50μm.A Multipotentdifferentiation of keloid-derived sphere colonies into different lineages of neural cells, adipocytes, and osteocytesas determined by immunofluorescencestaining with specific neural cell markers, Oil Red O and von Kossastaining. Scale bars, 50μm. The results are representative of at lease five independent experiments.

Figure 11. Expression of IL-6 receptor (IL-6R) inkeloids(Kl, K2) and matched normal skins (Nl, N2).A, Paraffin-embedded sections of keloid and the matched normal skin were immunostained with a specific antibody for human IL-6R or an isotype-matched controlIgG. Scale bars, δOμm.S, Western blot analysis of IL-6R in tissue lysates. C, Flow cytometricanalysis of IL-6R and IL- 17R expression in cultured SKPs or KPCs. The results are representative of five independent experiments.

Figure 12. Effect of IL-6 on the expression of SSEA-4 and BrdU incorporation in SKPs and KPCs.A,Cells were stimulated with 20ng/ml IL-6 for 24h followed byimmunostainedwith antibodies for SSEA-4 and FIT C -conjugated secondary antibody and analyzed by flowcytometry. B and C,Effects of IL-6 and IL- 17 neutralizing antibodies on cell viability and proliferation in SKPs andKPCs. Cells were treated for 24h with 5μg/ml of neutralizing antibodies for human IL-6, or IL- 17, or both in the absence of IL-6 and IL- 17, whereby anisotype-matched normal mice antibody (mlgG) was used as negative controls. Cell viability and proliferation were determined by MTT (B) andBrdUincorporation assay (C), respectively. The results are representative of at least five independent experiments (mean ±SEM). #, no significant difference; * P< 0.05, as compared with non- treatment control. Figure 13. Schemed inflammatory niche-driven benign tumor growth model. Under the chronic inflammatorymicroenvironment,keloid- derived precursor cells (KPCs) are persistently interacted with inflammatory cells and stimulated by enriched milieu of pro-inflammatory mediators, specifically IL-6, and then acquired a benign tumor-like stem cell phenotype characterized by moderately increasedtelomeraseactivity, and consequently, an increasedproliferativecapacity, whereby the increased IL-17 continuously drives this process by augmenting the production of IL-6 byKPCs, thus leading to the overgrowth ofkeloid benign tumor. DESCRIPTION OF THE INVENTION Alteration in stem cell niche is likely to contribute to tumorigenesis; however, the concept of niche promoted benign tumor growth remains to be explored. Here we used keloid, a benign tumor characterized by an exuberant fibroproliferative dermal growth unique to human skin, as a model to test our hypothesis that the altered niche in keloids, predominantly inflammatory, contributes to the acquirement of a benign tumor-like stem cell phenotype of keloid derived precursor cells (EDPCs) and in vivo modification of the "pathological" stem cell niche might be a novel therapy for keloid and other mesenchynimal benign tumors.

The following examples are intended to illustrate, but not to limit, the scope of the invention. While such examples are typical of those that might be used, other procedures known to those skilled in the art may alternatively be utilized. Indeed, those of ordinary skill in the art can readily envision and produce further embodiments, based on the teachings herein, without undue experimentation. Materials and Methods Antibodies and reagents AU antibodies used in this study are listed in Supplementary Materials and Methods (Table Sl). Recombinant human interleukin-6 and interleukin-17A were purchased from PeproTech Inc. (Rocky Hills, NJ, USA). All chemical reagents were analytical grade and obtained from Sigma-Aldrich (St. Louis, MO). Animals

C57BL/6 mice were purchased from Jackson Lab. Immunocompromised mice (bg-nu/nu-xid) were purchased from Harlan Sprague Dawley Inc. (Indianapolis, IN) and were used for in vivo transplantation experiments under the approved animal protocol of University of Southern California (USC #10874). Cell isolation and culture

Keloid and matched peripheral normal skin tissues were obtained from the same patient donor and were used for isolation of skin derived precursor cells (SKPs). At least 5 sets of keloid derived precursor cells (KPCs) with appropriate matched SKPs were used in all experiments described. SKPs were also derived from normal skin tissues from other donors. Tissues were treated aseptically and cut into 5-6 mm long pieces and incubated in sterile phosphate-buffered solution (PBS) containing 3mg/mL dispase (Sigma) overnight at 4°C. The epidermis was then manually stripped off and the dermal portion was cut into 1-mm³ pieces and digested in sterile PBS containing 4mg/mL collagenase I (Worthington Biochemical Corporation, Lakewood, NJ) for 2h at 37°C [50, 51], filtered through a 70μm cell strainer (Falcon, Franklin Lakes, NJ), and the isolated cells were cultured under two different conditions. For sphere -forming culture [50, 52, 53], cells were seeded in uncoated dishes with growth medium, Dulbecco's minimum essential medium with low glucose (DMEM- LG)/F12 (3: 1) (Invitrogen, Carlsbad, CA) supplemented with 40 ng/ml fibroblast growth factor (FGF-2), 20 ng/ml epidermal growth factor (EGF) (Chemicon, Billerica, MA)₁ B27, 1 μg/ml fungizone and 100 U/ml penicillin/lOOμg/ml streptomycin (Invitrogen). After 2-4 weeks, spheres were formed and further sub-cultured in 60 mm ultra-low culture dishes (Corning Inc., Corning, NY). For cell attachment or MSC culture, cells were cultured as described previously with some modifications [54], Briefly, resuspended cells were plated on non-treated 10-cm Petri dishes (VWR Scientific Products, Willard, OH) with minimum essential medium (MEM: Invitrogen) containing 10% fetal bovine serum (FBS: Clontech Laboratories, Inc., Mountain View, CA), 100 U/ml penicillin, 100 μg/ml streptomycin (Invitrogen), 2 mM L-glutamine, 100 mM non-essential amino acid (NEAA), and 550 μM 2-mercaptoethanol (2-ME; Sigma -Aldrich), and cultured at 37°C in a humidified tissue-culture incubator with 5% CO2 and 95% O2. The confluent cells were passaged with 0.05% trypsin containing ImM EDTA, and continuously subcultured in the complete growth medium.

Colony forming unit fibroblasts (CFU-F) assay

The CFU-F assay was performed as previously described [55, 56]. Cell aggregates containing more than 50 cells were counted as colonies using a dissecting microscope. The CFU-F assay was repeated in 5 independent experiments. Single cell cloning

Precursor cells isolated from keloid tissues and matched peripheral normal skin samples of different donors were seeded in non-coated tissue culture 96-well plates (Falcon) at a concentration of 2 cells/ml (200 μl/well, at least 4 plates/donor). The plates were screened for presence of single cell colony while wells contained more than two colonies were excluded from further analysis. Wells containing a single cell were allowed to reach confLuency, transferred to 24-well dishes, and further expanded in the growth medium [54] . Population doubling assay

Clonal dermal precursor cells at each passage (P2, P5, PlO and P20) were seeded at 1.OxIO³ cells in 35-mm dishes in MSC medium for each period (0, 2, 4, 6, 8, 10 days). Cells were suspended with 0.05% trypsin- EDTA and cell number was determined by hemacytometer. Population doubling time (PDT) was calculated with the formula, PDT=(t- to)*lg2/lg(N/No) (No and N represent the cell numbers at the time to and t, respectively). Meanwhile, the accumulated population doublings were determined and calculated according to the standard 3T3 protocol as described previously [57], Multipotent differentiation

We tested the in vitro multi- differentiation capabilities of dermal precursor cells including osteogenesis, adipogenesis and neural cell differentiation as described in Supplementary Methods. Stimulation of cells with IL-6 or IL-I 7 KPCs and SKPs at 70-80% confluence were serum starved (in medium containing 1% FBS) for 24 hours followed by exposure to different concentrations of IL-6 or IL-17 (R & D Systems) for another 24 hours. Under certain conditions, the starved cells were pretreated with different concentrations of specific neutralizing antibodies for human IL-6 or IL-17 or an isotype-matched control IgG (R & D Systems) for 1 hour followed by stimulation with IL-6 or IL-17 for 24 hours. Then whole cell lysates or total RNA were extracted from cells and submitted to Western blot and RT-PCR analysis, respectively. Cell proliferation assay The proliferation of dermal precursor cells was assessed by 5-bromo-

2-deoxyuridine (BrdU) incorporation using a BrdU staining kit (Invitrogen) as previously described [52]. To quantify cell proliferation capacity, ten representative images were used to enumerate BrdU-positive cells and quantitated as a percentage of BrdU-positive cells over total nucleated cells. Flow cytometric analysis

KPCs and SKPs at 70-80% confluence were serum-starved (medium containing 1% FBS) for 24 hours followed by exposure to different concentrations of IL-6 or IL-17 (R & D Systems). Under certain conditions, to clarify the role of IL-17/IL6 axis in the regulation of KPC functions, the serum-starved cells were pretreated with different concentrations of specific neutralizing antibodies for human IL-6 or IL-17 or an isotype-matched control IgG (R & D Systems) for 1 hour followed by stimulation with IL-6 or IL- 17 for 24 hours. Then whole cell lysates or total RNA were extracted and subjected to Western blot and RT-PCR analysis, respectively [55,56]. Reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR) Total RNA was isolated from cultured cells using TRIZOL^® Reagent

(Invitrogen). RT-PCR was carried out using the One-step RT-PCR Kit (QIAGEN, Valencia, CA), whereby the number of amplification cycles and the amplifying condition for individual target genes were determined to be in a linear range of amplification. To confirm RT-PCR results in some experiments, qPCR was performed using the iScript one-step RT-PCR kit with SYBR Green (BioRad, Hercules, CA) according to the manufacturer's instructions. As an internal control, levels of β-actin were determined in parallel with target genes. All primers (Table S 2) were synthesized at the Core Facility at Norris Comprehensive Cancer Center, USC. We ste r n blottin g

We performed electrophoresis of protein extracts prepared from cultured cells or tissue lysates followed by immunoblotting as previously described [51]. Blots were incubated with primary antibodies at a dilution of 1:1000-1:2000 followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (PIERCE, Rockford, IL), and visualized by the enhanced chemiluminescence light method (PIERCE). For standardization, blots were re-probed with mouse antibody to human B-actin. Cytokine antibody array

To analyze the cytokine expression profile of tissue lysates we utilized the RayBio Human Cytokine Antibody Array 3 (RayBiotech, Inc., Norcross, GA), which allows the detection of 42 cytokines, chemokines and growth factors in one experiment (Figure 3A) following manufacturer's protocol. Teloraerase activity and telomeric length assay The telomerase activity was measured using the telomere repeat amplification protocol (TRAP) TeIoTAGGG telomerase PCR ELISA kit (Roche Diagnostics, Indianapolis, IN). The telomerase-mediated elongated product was detected by hybridization to digoxigenin-labeled probes and enzyme activity quantified by photometric enzyme immunoassay. Enzyme-linked immunoassay (ELISA)

The level of IL-6 and IL-17 in tissue and cell lysates was detected using human IL-6 ELISA Ready-SET-Go (eBioscience, San Diego, CA) and human or mouse IL-17 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN), respectively, following the manufacturers' instructions, In vivo transplantation

Transplantation studies were carried out using KPCs and matched SKPs derived from five different donors, and each transplantation was done in triplicate (n=3). Approximately 2,0 x 10⁶ stem cells mixed with 40 mg of hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic powder (Zimmer Inc., Warsaw, IN), or Gelfoam (3mm x 3mm x 2mm, Pharmacia, Piscataway, NJ), were subcutaneously transplanted into the dorsal surface of 8-10-week-old female immunocompromised mice as previously described [55, 56]. For the hydrogel carrier transplant, we mixed 2.0 x 10⁶ cells with Heprasil™ (CMHA-S, thiol- modified hyaluronic acid with thiol-modified heparin) and Gelin-S™ (GTN-DTPH, thiol-modified gelatin) (1: 1) with or without IL-6, and then combined with Extralink™ (PEGDA, polyethylene glycol diacrylate) (4: 1) (Glycosan Biosystems, Salt Lake City, UT). The immobilized heparin in Extracel™-HP in the form of Heprasil™ mimics heparin sulfate proteoglycans normally present in the extracellular matrix, increases the avidity of growth factor binding non-covalently to the polymer network, protects the growth factors from proteolysis and maintains their bioactive state while sequestering and slowly releasing them from the hydrogel over several weeks (Glycosan Biosystems) [58, 59], The mixtures were immediately injected subcutaneously into the dorsal surface of 8-10- week-old female immunocompromised mice.

To further investigate the role of IL-6 in the initiation of KPC- generated transplants, KPCs mixed with hydrogel with or without human IL-6 neutralizing antibody (lOμg/ml) were injected subcutaneously into immunocompromised mice for 8 weeks. On the other hand, to investigate the role of IL-6 in the maintenance of KPC-generated transplants, KPCs mixed with hydrogel were injected subcutaneously into immunocompromised mice for 4 weeks, followed by intra- Ie sional injection of IL-6 neutralizing antibody twice a week (5μg/time) for another 5 weeks. In all cases, an isotype-matched normal mice IgG was used as negative control.

Adaptation of activated CD4⁺CD25- T-lymphocytes in immunocompromised mice

Spleens were harvested from C57BL/6 mice for isolation of CD4⁺CD25- T-lymphocytes using MidiMACS separator (Miltenyi Biotec) and mouse CD4⁺CD25⁺ T lymphocyte isolation kit (Miltenyi Biotec) following manufacturer's instruction. The purity of the CD4⁺CD25" T cells was >95%. The cells were seeded at 1 x 10⁶/well on 24-well multi-plates and cultured in RPMI- 1640 medium containing 10% FBS₁ 50 μM 2ME, 10 mM HBPES, 1 mM sodium pyruvate, 1% NEAA, 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin followed by stimulation with 5 μg/ml plate-bounded anti-CD3 antibody (BD Biosciences) and 2 μg/ml soluble anti- CD28 (BD Biosciences). After three days, activated cells were collected and washed with normal saline [60]. 1 x 10⁶/mouse of the activated CD4⁺CD25- T lymphocytes were injected into immunocompromised mice through tail vein two days before KPCs mixed with hydrogel with or without IL-6 were transplanted subcutaneously. In parallel, transplants in mice without infusion of T cells were used as controls. Histology and immunohistochemistry Keloid and matched peripheral normal skin tissues, normal skin and normal scar tissues from both African-American and Caucasian patient donors, and transplant samples were fixed with 10% formalin in PBS or embedded in O. CT. compound (Sakura, Tokyo, Japan) and snap frozen in liquid nitrogen. Procedures for immunohistochemical studies were described in Supplementary Methods, immunofluorescence studies

4% paraformaldehyde-fixed cultured cells and sections of normal and keloid tissue samples were immunolabeled with specific primary antibodies followed by FITC- and/or PE-conjugated secondary antibodies (BD Biosciences). After the nuclei were stained with 4', 6-diamidino-2- phenylindole (DAPI) the samples were observed under a confocal microscope. Isotype-matched control antibodies (Invitrogen) were used as negative controls. Positive signals in at least 5 random fields were visualized and counted. Statistical analysis

All data are expressed as mean ± SEM from at least five independent experiments. Differences between experimental and control groups were analyzed by two-tailed unpaired Student's t-test using SPSS. P-values less than 0.05 were considered statistically significant. SUPPLEMENTARY METHODS Multipotent differentiation of dermal derived precursor cells

Osteogenic differentiation: SKPs and KPCs were plated at 5 x 10⁵ cells/well in 6-well plate in mesenchymal stem cell (MSC) growth medium, allowed to adhere overnight, and replaced with Osteogenic Induction Medium (PT-3002, Cambrex, Charles City, IA) supplemented with dexamethasone, L-glutamine, ascorbic acid, and β-glycerophosphate. After 4-5 weeks, the in vitro mineralization was assayed by Alizarin red S (Sigma-Aldrich) staining and quantified by acetic acid extraction method [79]. Under some conditions, von Kossa staining was used to identify extracellular mineralized matrix.

Adipogenic differentiation: SKPs and KPCs were plated at 5 x 10⁵ cells/well in 6-well plate in MSC growth medium, allowed to adhere overnight, and replaced with adipogenic induction medium supplemented with 10 μM human insulin, 1 μM dexamethasone, 200 μM indomethacin, and 0.5 mM 3-isobutyl-l-methylxanthine (Sigma-Aldrich, St Louis, MO). Oil Red O staining was performed to detect intracellular lipid vacuoles characteristic of adipocytes, and the dye content was quantified by isopropanol elution (5min shaking) and spectrophotometry at 510 nm [80].

Neuronal differentiation: Single cells were dissociated from keloid- derived precursor cell (KPCs) spheres and plated at 5 x 10³ cells/well in 8- well chamber slides (Nalge Nunc, Rochester, NY) coated with poly-d- lysine/laminin and cultured in DMEM/F12 (3:1) (Invitrogen, Carlsbad, CA) supplemented with 40ng/ml FGF-2 (Chemicon, Billerica, MA) and 10% FBS (Invitrogen) for 5~7 days. For neurogenic induction cells were cultured for another 5-7 days in the same medium without FGF-2, but with the addition of 10 ng/ml nerve growth factor (NGF), 10 ng/ml brain-derived neurotrophic factor (BDNF), and 10 ng/ml NT-3 (Peprotech, Rocky Hill, NJ). To differentiate KPCs into Schwann cells, dissociated spheres were cultured in DMEM/F12 (3:1) containing 10% FBS for 1 week, then switched to the same medium supplemented with 4μM forskolin (Sigma). In all experiments, cells were induced to differentiate for 2-3 weeks, with 50% of the medium changed every 3-4 days. Karyotyping

Karyotyping was performed by the Cytogenetics Core Laboratory of City of Hope and Beckman Research Institute

(http : //w w w . citvofhop e . or g/r e se ar ch/supp ort/cvtoge ne tics/Pages/def ault. aspx ) (Duarte, CA). SKPs and KPCs at passage 10 derived from the same patient were cultured as described in Materials and Methods until near confluency. Metaphase spreads were prepared and chromosome analysis was performed according to standard procedures. Serial transplantation

To further confirm the in vivo differentiation ability of KPCs and SKPs, we performed serial transplantation as well as transplantation using a limiting dilution assay. For serial transplantation, 2 x 10⁶ KPCs were transplanted subcutaneously into immunoconipressed mice. After 4 weeks, the transplants were harvested and recovered single cells were expanded and re-transplanted into immunocompressed mice for another 4 weeks. Meanwhile, various numbers of KPCs and SKPs at 2 x 10<\ 1 x 10⁶, 5 x 10⁵ _; 1 x 10⁵, 1 x 10⁴, 1 x 10³, and 1 x 10² were transplanted subcutaneously into immunocompromised mice, and 8 weeks later the transplants were harvested. Immunohistochemical studies The paraffin- or frozen sections were incubated with primary antibodies and detected using the universal immunoperoxidase (HRP) ABC kit (Vector, Burlingamβj CA). They were counterstained with hematoxylin. Isotype-matched control antibodies (Invitrogen) were used as negative controls. For histological study, paraffin sections were stained with hematoxylin and eosin (H&E). To quantify Oct-4 and SSEA-4 expression, sections were visualized using a 4OX objective, and positively stained cells in 5 random high-power fields (HPF) were counted and expressed as percentage of total cells [81]. Electron microscopy

The samples of KPC and SKP transplants were fixed in 2.5% glutaraldehyde and embedded in epoxy resin. The ultra-thin sections (70 μm) were stained with uranyl acetate and lead citrate and observed under a transmission electron microscopy. RESULTS

Keloid contains benign tumor-like stem cells

Aside from the characteristic histological features of abundant ECM stroma, specifically large collagen bundles (Figure 8B), hyper- cellular and inflammatory states, the keloid tissue harbors an elevated level of Octamer- 4 (Oct-4) and stage specific embryonic antigen-4 (SSEA-4) positive signals randomly dispersed in the reticular dermis as compared to matched normal skin of the same donor (Figure IA; Figure 8C). The relatively increased expression of two major markers for embryonic and mesenchymal stem cells [50, 61, 62], Oct-4 and SSEA-4, in keloid tissues as compared to matched normal skin was further confirmed by Western blot (Figure IB), suggesting that keloid may harbor more postnatal stem cells than normal skin.

Next, we isolated a population of adult stem cells from keloid dermis, named keloid derived precursor cells (KPCs), from 5 independent donors and assess their colony formation and proliferation. Normal skin-derived precursor cells, termed SKPs [50], were derived from matched normal skin of the same donor. Colony formation was observed on day 3 (Figure 8D) in 6-8 % of KPCs whereas only 2-4% in SKPs (Figure 1C), suggesting higher colony-forming capabilities of the tumor-like stem cells in keloids. Single colony formation was further confirmed using diluting seeding density at 2 cells/ml (200 μl per well) (Figure 8E). At early passages (less than passage 5), both KPCs and SKPs exhibited similar doubling time and cumulative population doublings; however, at a later passage, KPCs showed a relatively increased growth rate compared to SKPs, corresponding to an increase in cumulative population doublings and a reduction in doubling time (Figure ID; Figure 8F). G-banding analysis demonstrated normal karyotype with no cytogenetic abnormalities in both SKPs and KPCs (Figure 8G), suggesting the maintenance of chromosomal stability in these derived stem cells. These results suggest that keloid-derived stem cells maintain a relatively higher proliferative rate than their normal skin counterparts, and this inherent rapid growth appears unrelated to mutational changes observed in malignant tumor. Up to date, efforts have been made to develop a cell-surface antigen profile for better purification and identification of mesenchymal stem cells (MSCs) from different tissue sources [3], There is a consensus that human MSCs rarely express markers of hematopoietic cells such as CD45 and CD34 [3, 61, 63]. Several positive markers such as Stro-1, CD73, and CD 106 were considered the most useful and relatively specific markers for MSCs [3], but others including CD90/Thy-l were relatively non-specific, although frequently expressed on MSCs from different tissues [3, 61, 63], To characterize stem cell phenotypic markers of KPCs using flow cytometry, we observed that a high percentage of both KPCs and SKPs expressed CD90 at passage 2-6 (Figure IE). However, only 11% and 46.4% of KPCs expressed CD29 and CD73, respectively, as compared to 77.6% for CD29 and 77.1% for CD73 in SKPs (Figure IE). Interestingly, the percentage of KPCs expressing Stro-1 (19.1%) and CD105 (4.5%) was consistently higher than that of SKPs (Figure IE). The differential expression of mesenchymal stem cell marker profiles suggests KPCs are uniquely distinct from SKPs.

We next examined several stem cell specific genes expression using quantitative real-time PCR (qPCR). KPCs expressed relatively higher levels of Oct-4, Rex-1, Nanog, and human telomerase reverse transcriptase (hTERT) mRNAs than SKPs (Figure IF). In addition, comparative analysis of telomerase activity using TeIo TAGGG telomerase PCR ELISA showed that KPCs displayed an inherently higher telomerase activity (5-6 folds) as compared to normal skin stem cells (Figure IG). Unlike cancer, both KPCs and SKPs showed significantly lower level of telomerase activities than cancer cells (Figure IG), and gradually decreased with increasing cell passages (Figure IH) suggesting a tighter growth regulation. Taken together, KPCs differ from SKPs, and display distinct surface markers, functional properties, colony formation and proliferative index, resembling benign tumor-like properties.

Keloid-derived precursor cells are capable of multiple differentiation

The multi- differentiation potential of KPCs was determined and compared to that of SKPs. Under adipogenic and osteogenic induction conditions, both SKPs and KPCs could differentiate into adipocytes and osteoblasts as determined by Oil Red O staining (Figure 9A) and by Alizarin Red S staining (Figure 9D), respectively. Adipogenic differentiation was further confirmed by the increased expression of specific adipogenic markers including peroxisome proliferators-activated receptor γ2 (PPARγ2), αP2 and lipoprotein lipase (LPL) as determined by RT-PCR (Figure 9C). Similarly, the osteogenic induction of KPCs and SKPs was further supported by the increased expression of osteocalcin, an osteogenic marker (Figure 9F). In addition, quantification of the Oil Red O and Alizarin Red S (Figures 9B and 9E) staining using 5 random fields as well as RT-PCR results (Figures 9C and 9F) showed a moderate increase in the expression of adipogenic and osteogenic markers in differentiated KPCs compared to differentiated SKPs. However, further evidence is needed to determine whether KPCs have a relatively stronger capability to differentiate toward adipogenesis and osteogenesis as compared to SKPs. Meanwhile, we demonstrated that keloid-derived sphere colonies (Figure 10) were capable of multipotent differentiation into distinct lineages including mesoderm- derived adipocytes, osteoblasts and smooth muscle cell-like cells, and ectoderm -derived different types of neural cells (Figure 10D). These findings consistent with previous stem cell properties described in rodent and mammalian skin-derived precursors or SKPs [50, 52, 53] indicate that keloid derived stem cells may represent an active form of SKPs.

KPCs are capable of regenerating connective tissues after in vivo transplantation

To explore the in vivo regenerative capability, the expanded KPCs and SKPs (2 xlO⁶) were subcutaneously transplanted with Gelfoam as a carrier in immunocompromised mice. Similar transplants were carried out using human bone marrow mesenchymal stem cells (hBMMSCs) as another source of stem cells. As expected, hBMMSCs were not capable of regenerating tissue up to 8 weeks. However, KPCs and SKPs from different donors consistently regenerated connective tissue-like transplants (5 out of 5 mice), with a more rapidly growing tumor-like mass using KPCs as compared to SKPs (Figure 2A). The human origin of cellular components of the transplants was confirmed by immunostaining with specific antibodies for human mitochondria (Figure 2B). Histological features of the transplants resembled early connective tissue phenotype including presence of type I collagen fibrils, however, the characteristic keloid-like thick collagen bundles were not observed (Figures 2B and 2C). Next, to assess the in vivo bone regenerative capability, KPCs were implanted subcutaneously with hydroxyapatite/tricalcium phosphate (HA/TCP) as a carrier in immunocompromised mice for 8 weeks. Histological analyses revealed calcified nodules in both KPCs and SKPs generated transplants (Figure 2D).

To further confirm that the isolated KPCs and SKPs are truly stem cells, we carried out a series of transplantation using limiting cell density dilution. As shown in Table S4, KPCs exhibited stronger capability for tumor xenograft formation at a minimal density of 1 x 10⁵cells after transplantation for 8 weeks as compared to their counterparts, SKPs. We next performed serial transplantation with hydrogel as a carrier using 2 x 106 KPCs subcutaneously transplanted into immunocompromised mice. At 4 weeks post-primary transplantation, the transplants were harvested and digested single cells were re-transplanted into immunocompromised mice for another 4 weeks. Our results indicated that KPCs recovered from secondary transplants maintained the in υiυo ability to form connective-like tissues (Figure 2E). These results were unique and particularly striking, as KPCs transplanted to a heterologous environment were capable of proliferating and differentiating into scar-like connective tissues expressing type I collagen. However, we were not successful in replicating the human keloid tumor using the current transplant model, suggesting that KPCs are not the sole contributor to the exuberant scar growth. Therefore, we hypothesize that, aside from KPCs, the environmental niche where KPCs reside, is capable to drive stem cell proliferation and differentiation, and therefore regulates the tumor-like keloid growth. Elevated IL-17 and IL-6 coordinately contribute to the unique inflammatory niche of KPCs

To further explore the concept of ^' niche regulated keloid fibroproliferative growth we next attempted to define the distinct "pathological" niche of keloid by screening a panel of candidate inflammatory cytokines utilizing fresh keloid tissues from different donors. Using a focused human cytokine array we found that the levels of several mediators, including growth-regulated IL-6, oncogene alpha (GROα), IL-IB, MlP-lδ, RANTES, stem cell factor (SCF), TGF-βl, TNF-α, angiogenin II, vascular endothelial growth factor (VEGF) and platelet- derived growth factor beta homodimer (PDGP-BB), were increased by more than 2-folds in keloid tissues as compared to matched normal skin tissues (Figure 3A; Table S3). Particularly, an elevated level of IL-6, a critical cytokine of the T_H2 cells, and its receptor IL-6R, were detected in all keloid tissues screened and further confirmed by immunohistochemical studies (Figure 3C; Figure HA) and Western blot analysis (Figure 3F; Figure 11B). Both IL-6 and IL-6R were expressed in SKPs and KPCs, with a relatively higher level in KPCs as compared to the matched SKPs, as determined by Western blot and RT-PCR (Figures 3G and 3H).

Another important inflammatory cytokine of the TH17 cells [33-36], IL- 17, was abundantly expressed in keloid tissues as demonstrated by ELISA (Figure 3B) and further confirmed by immunohistochemical staining (Figure 3C) and Western blot analysis (Figure 3E). The increased expression of IL-17 in keloid tissues was discernible around T-cell receptor CD3-positive cells as determined by dual-color immunofluorescence staining (Figure 3D). IL-17 has been reported to be produced almost exclusively by activated T cells, but its receptor is widely expressed in various cell types [37], Herein, we observed an elevated expression of IL- 17R in KPCs as compared to SKPs (Figure 31). However, the expression of IL-17 was undetectable in both KPCs and SKPs. Taken together, these findings suggest that IL-6 and IL-17 are abundant components of the unique inflammatory niche of keloid and may contribute to the regulation of self-renewal and differentiation of keloid stem cells.

IL-17 is involved in the regulation of hTERT expression in KPCs via coordinating IL-6 autocrine/paracrine loop

IL-6 plays an important role in regulating self-renewal and differentiation of stem cells [16, 17, 20], as well as tumor growth [20-22]. Our data showed that IL-6 stimulated BrdU incorporation in both KPCs and SKPs (Figure 4A). Exposure of KPCs and SKPs to exogenous IL-6 resulted in a dose -dependent increase in Oct-4 expression at both mRNA and protein levels (Figures 4B and 4C). In parallel studies, we found that incubation with IL-6 enhanced telomerase (hTERT) expression and enzyme activities in both KPCs and SKPs (Figures 4D, 4E and 4F). The basal level of hTERT was always higher in KPCs as compared to SKPs. Since KPCs constitutively expressed a higher level of IL-6 and IL-6R than SKPs (Figures 3G and 3H), we next tested whether the intrinsically secreted IL-6 might contribute to the elevated telomerase expression and activity in KPCs. Our data showed that treatment with IL-6 neutralizing antibodies suppressed hTERT expression in both KPCs and SKPs (Figures 4G and 4H) in a dose -dependent manner. However, IL-6 showed no obvious effects on SSEA-4 expression in both SK-Ps and KPCs as determined by flow cytometry (Figure 12A). Taken together, these data suggest that ΪL-6 plays a critical role in the regulation of self-renewal and proliferation of KPCs by regulating essential functional genes such as hTERT and Oct-4.

Most recently, studies have shown that IL-6 participates in the lineage commitment of pathogenic IL-17-producing T helper cells (TH17 cells) from naϊve T cells [64-66]. On the other hand, IL- 17 induces the secretion of IL-6 in a variety of cells including fibroblasts [39, 45] and epithelial cells [46]. We then explored the interaction between IL-6 and IL- 17 and determine whether the IL-17/IL-6 axis contributes to the regulation of the keloid niche. Our results showed that incubation with IL- 17 enhanced IL-6 expression in both SKPs and KPCs (Figure 5A). However, exposure of KPCs to exogenous IL-6 failed to stimulate IL- 17 secretion. Similar to IL-6, IL- 17 enhanced hTERT and Oct-4 expression in both SKPs and KPCs (Figure 5B). Treatment with IL-6 neutralizing antibody decreased the basal level of hTERT and Oct-4, and in conjunction with IL- 17 antibody only slightly augmented the blockade of hTERT and Oct-4 expression in both SKPs and KPCs (Figure 5C). To further delineate the relationship of IL-6 and IL- 17 in the regulation of hTERT and Oct-4 expression in SKPs and KPCs, we pretreated cells with neutralizing antibody for either IL- 17, or IL-6, or both followed by IL- 17 exposure. Our results indicated that IL-17-induced hTERT and Oct-4 expression was abolished by pretreatment with neutralizing antibody for either IL- 17, or IL-6, or both (Figure 5D). However, IL-6-induced up-regulation of hTERT and Oct-4 expression was only negated by pre-treatment with IL-6 neutralizing antibody, not by IL- 17 neutralizing antibody (Figure 5E), Meanwhile, we examined whether treatment with IL-6 and IL- 17 neutralizing antibodies had any effects on cell proliferation and cell viability of SKPs and KPCs. Our results showed that treatment with IL-6, but not IL- 17, neutralizing antibodies moderately inhibited BrdU incorporation (P<0.05) (Figure 12C), but had no obvious effects on cell viability (Figure 12B). We observed no apparent morphologic change characteristic of apoptosis after treatment with IL-6 and/or IL-17 neutralizing antibodies (data not shown). In all experiments, an isotype- matched antibody (mouse IgG) was used as negative controls. Taken together, these results suggest that IL-17 induces hTERT and Oct-4 expression in SKPs and KPCs by orchestrating the downstream IL-6 autocrine/paracrine loop.

IL-6/IL-17 axis contributes to the fibroproliferative growth of KPCs- generated transplants In order to recapitulate the distinct "pathological" niche of keloid, we engineered the in vitro inflammatory niche by incorporating IL-6 non- covalently into the hydrogel carrier. KPCs and SKPs (2 x 10⁶) were mixed with IL-6-linked carrier and immediately injected subcutaneously on the dorsal surface of immunocompromised mice. Our results showed that KPCs- generated transplant grew faster than that from SKPs, and the presence of IL-6 in hydrogel carrier significantly promoted the in vivo growth of both KPCs and SKPs (Figure 6A). Histological analyses revealed significant increase in cellular components and type I collagen expression in all transplants using IL-6-retained hydrogel as compared to hydrogel only (Figures 6B and 6C). The collagen structure of IL-6 treated transplant was more fibrillar and less amorphous. Importantly, abundant keloid-like thick collagen fibrils were observed in KPC transplants treated with IL-6 as demonstrated by histological and immunohistological staining (Figures 6B and 6C), and electron microscopy (Figure 6D). The unique pattern of excessive intracellular and extracellular accumulation of collagen was seen in KPC transplants (Figure 8B; Figure 6D). Meanwhile, we demonstrated that transplants generated from KPCs and SKPs in the presence of IL-6 expressed higher levels of Oct-4 and hTERT, as well as an elevated hTERT enzyme activity, as compared to control (Figures 6E, 6F and 6G). The increased proliferation of dermal stem cells by IL-6 was further confirmed by immunohistochemical staining with antibody for human PCNA (proliferating cell nuclear antigen) (Figure 6H). These results provided further evidence that IL-6, as a downstream target of IL- 17 and an essential component of the unique inflammatory niche of keloid, plays a critical role in the regulation of self-renewal and differentiation of keloid stem cells. To explore whether IL-6 plays a role in the initiation of KPC- generated transplants, KPCs mixed with hydrogel with or without human IL-6 neutralizing antibody were injected subcutaneously into immunocompromised mice whereas isotype-matched antibody (mouse IgG) was used as negative control. Our results indicated that treatment with IL- 6 neutralizing antibody significantly suppressed the in vivo growth of KPC- generated transplants (Figure 61). We next examined whether IL-6 is capable of maintaining the growth of KPC-generated transplants. KPCs mixed with hydrogel were injected subcutaneously into immunocompromised mice. Four weeks later, IL-6 neutralizing antibody was injected locally into the transplant, twice a week (5μg/injection) for 4 weeks. Our results showed that intra-lesional injection with IL-6 neutralizing antibody significantly reduced the size of KPC-generated transplants (Figure 6J). These results provided further evidences that IL-6 plays an important role in both initiation and maintenance of KPC transplants, suggesting a potential anti-scarring therapeutic effect.

Since our KPC transplant studies were carried out in athymic nude (nu/nu) mouse which may be devoid of functional T lymphocytes [67], we transplanted KPCs using hydrogel with and without IL-6 into immunocompromised mice adapted by CD4⁺CD25- T-lymphocytes (Naϊve T) infusion. Our results showed that boosting T cell function in immunocompromised mice significantly promoted growth of KPC transplants, in the presence or absence of IL-6 (Figure 7A). Immunofluorescence studies showed an increased expression of IL- 17 by CD4 positive-T-lymphocytes, which was further enhanced by IL-6 treatment (Figure 7B). The increased IL-17 expression was further confirmed by ELISA assay (Figure 7C). All together, we have shown that the IL-6/IL-17 axis functions to maintain the highly elevated IL-6 level in keloid with abilities to regulate key stem cell functions such as hTERT and Oct-4 expression. Therefore, we have further confirmed that the distinct keloid niche constituted by the IL-17/IL-6 axis is essential in the regulation of KPCs proliferation and differentiation, therefore contributes to the formation of the benign keloid growth. Discussion

Unlike a more precise topographical organization of epithelial stem cells in skin epithelium, the dermal layer harbors Oct-4 and SSEA-4 positive cells that are randomly distributed in the reticular dermis, interspersed among thick ECM, as seen abundantly resided in keloid tissues. The relatively abundant expression of two major markers for embryonic and mesenchymal stem cells [50, 61, 62], Oct-4 and SSEA-4, in keloid as compared to normal skin tissues, suggests the presence of postnatal stem cells in pathologic scar and may contribute to its persistent growth.

In the last few decades, several groups have shown that multipotent precursor cells can be generated from different normal adult human tissues [61-63, 68, 69], and may share common biological properties, including the expression profile of various cell surface markers and embryonic transcription markers as well as their multipotent differentiation capabilities [3, 63, 68]. Thus, adult stem cells may lend an essential role in growth, homeostasis and regeneration of many tissues. However, up to date, knowledge of how stem cells contribute to pathologic diseases remains relatively unwrapped. We asked whether stem cells derived from a keloid benign tumor possess distinct stem cell properties as their normal skin counterparts previously described as skin-derived precursor cells or SKPs [50], and whether their unique niche contribute to the pathogenesis of the fibrotic disease in terms of proliferation and differentiation. Several groups have reported the isolation and characterization of SKPs, from normal rodent and human normal skin dermis [50, 52, 53] and recently, mesenchymal-like stem cells from keloid scars [70], However, to our knowledge, no study has shown whether normal skin-derived SKPs differ from precursor cells derived from their aberrant scars, specifically their unique stem cell properties, in vivo self-renewal and regenerative capacities, and more importantly, whether the immediate local pathological niche driven by the inflammatory cytokines affect growth and differentiation. In this study, we have isolated and characterized a population of keloid derived precursor cells (KPCs) that exhibit stem cell properties, including clonogenicity, multiple differentiation capacities, and self-renewal. We found that KPCs possessed proliferative potential, expressed higher levels of the pluripotent state-specific transcription factors Oct-4, Nanog, and Rex-1, and displayed increased telomerase activity as compared to SKPs derived from the same donor's normal skin. Based on these inherently distinct stem cell properties, KPCs may represent the benign tumor-like stem cell counterpart of SKPs which might contribute, at least in part, to the high proliferative state in keloid tumor. Despite the tumor-like growth behavior, KPCs displayed normal karyotype and absence of cytogenetic abnormalities, which is most likely explained by the effect of the surrounding inflammatory environment on regulating the benign tumor growth.

More importantly, accumulating evidence has pointed to the critical role of stem cell niche in the regulation of stem cell functions. The balance between proliferation- inhibiting and proliferation-promoting signals provided by the specialized niche microenvironment is the key to homeostatic regulation of stem cell maintenance versus tissue regeneration [5]. Therefore, any changes in the niche components will lead to alterations of stem cell functions. A decline in signaling from the niche that is essential to the maintenance of stem cells can lead to aging and even loss of stem cell number and function [71, 72], thus contributing to aging or degenerative diseases. On the other hand, deregulation or alteration of the niche by dominant proliferation-promoting signals may lead to overgrowth of resident stem cells, thus contributing to hyper-proliferative diseases, benign and malignant tumors [5]. Keloid, a fib ro -proliferative benign tumor of skin, is characterized by chronic inflammation, an increased infiltration

3] of inflammatory cells, an enriched milieu of cytokines and growth factors, and an abundant accumulation of ECM [48, 49, 51], thus providing a unique inflammatory niche for the resident stem cells. Using keloid as a niche-related disease model we explore the critical niche components and their role in the regulation of stem cell functions.

In consistent with previous reports [12, 13], we have demonstrated that IL-6, an inflammatory cytokine that plays a critical role in the pathogenesis of most fibrotic diseases [11] and recently, chronic inflammation-associated tumorigenesis of colitis associated cancer [18, 19], is significantly up-regulated in keloid scars versus their normal skin counterparts. Moreover, we found that KPCs constitutively express a higher level of IL-6 and IL-6R compared to SKPs. These findings lead us to hypothesize that the locally enriched IL-6 in keloid scars might constitute a major component of the inflammatory niche where KPCs reside and play a critical role in the regulation of KPC functions. To test our hypothesis, we investigated the effect of IL-6 on the expression of two important stem cell markers Oct-4 and telomerase in both KPCs and SKPs. Our results show that IL-6 can significantly stimulate the expression of both Oct-4 and telomerase in cultured KPCs and SKPs, and incubation with specific IL-6 neutralizing antibodies dramatically decreased the constitutive expression of telomerase in KPCs.

Since an animal model for keloid scar does not currently exist, we generated a keloid animal model using dermal derived precursor cells. To recapitulate the "pathological" niche of keloid, we utilized a newly introduced carrier Extracel-HPTM Hydrogel conjugated with IL-6 for the in vivo transplantation of KPCs [58, 59]. We have demonstrated that IL-6 stimulates the expression of Oct-4 and telomerase in vivo and significantly promotes the growth of KPCs and SKPs-generated transplants. More importantly, abundant keloid-like collagen fibrils were accumulated in transplants of KPCs in the presence of IL-6 that resemble the characteristic histological features of keloid scars. Using the current KPCs-generated transplant as a keloid model we attempted to treat with IL-6 neutralizing antibody using standard intra-lesional injection approach and showed significant reduction in keloid size. Furthermore, pre-treatment of KPCs with IL-6 neutralizing antibody significantly suppressed the in vivo growth of KPCs-generated transplant, suggesting a potential anti-scarring preventive and therapeutic effect. Taken together, these important findings support the notion that IL-6 serves a major component of the inflammatory niche in keloid scars and play a critical role in the acquirement of benign tumor-like stem cell phenotypes, therefore the benign tumor growth.

Another recently identified inflammatory cytokine, IL- 17, exclusively produced by activated T cells and whose receptor is widely expressed in many cell types [37], plays a pivotal role in several inflammatory diseases [33-36]. IL-6, in concert with TGF-β, induces TH 17 cell differentiation from naϊve T cells by orchestrating a series of "downstream" cytokine-dependent signaling pathways [64-66]. More importantly, IL-6 has been reported to function both upstream of IL- 17 and downstream of IL- 17, constituting a positive feedback loop that promotes autoimmune and allergic diseases [45, 46]. In this study, we have demonstrated for the first time to our knowledge that IL-17 is up-regulated in keloid tissues, and KPCs express relatively higher IL- 17R than normal skin-derived precursor cells. Furthermore, we demonstrated that blocking IL-6 abolished IL-17-induced up-regulation of hTERT and Oct-4 expression; however, blocking IL- 17 failed to attenuate IL-6-induced up-regulation of hTERT and Oct-4 expression in KPCs. These findings support our hypothesis that elevated IL-6 and TGF-β in keloids promote differentiation of TH17 cells, which in turn secretes more IL-17 to further enhance the release of IL-6 from keloid stem cells or other stromal cells. The IL-17-triggered positive-feedback loop via autocrine/paracrine IL- 6 induction constitutes the "pathological" niche or altered inflammatory niche that drives KPCs toward proliferation, and thus tumor-like keloid growth (Figure 13). However, it is important to emphasize that despite the tumor-like growth behavior, KPCs were distinct from cancer cells or cancer stem-like cells (20) in that KPCs displayed a normal karyotype and an absence of cytogenetic abnormalities. Since inflammation plays a critical role in tumorigenesis (18, 19), longer in υiυo experimental observation, up to 2 months in our study, was carried out to determine the long-term effects of inflammatory niche components, specifically the IL-17/IL-6 axis, on the acquired properties of KPCs, including their replicative/proliferative capabilities, and cellular transformation. Due to the limitation of the biological activity of the hydrogel-released growth factors, we were not able to prolong the study beyond the above time point. Further studies will be needed to determine the effect of chronic inflammation on potential tumorigenesis. In addition to providing proliferation and differentiation signals, stem cell niche also provides homing signals to recruit bone marrow MSCs to sites of injury and inflammation [3, 5], whereby a variety of inflammatory mediators, including hypoxia, reactive oxygen species (ROS), inflammatory chemokines and cytokines, alone with tissue damage- produced by-products, may trigger the migration of BMMSCs [73]. For example, several studies have demonstrated that BMMSCs are preferentially recruited to tumor microenvironment characteristic of chronic inflammation [74, 75], wherein they become activated and acquire cancer-associated fibroblast (CAF) phenotype by differentiation after prolonged exposure to the unique cancer microenvironment and subsequently promote tumor growth and metastasis through contributing to the production of pro- angiogenic and tumor-stimulating paracrine factors [76, 77], Similarly, a recent study by Akino et al has shown that keloid- derived fibroblasts are able to induce human BMMSCs chemoattraction toward keloid cells, and these human MSCs when cocultured with keloid fibroblasts tend to differentiate into myofibroblasts with abundant myofibers, rough endoplasmic reticulae and the secretion of collagen bundles, albeit no similar effects were observed with normal fibroblasts [78]. These findings suggest that the unique keloid microenvironment or niche may preferentially recruit bone marrow MSCs, where they become activated and differentiated into myofibroblasts, thus contributing to keloid formation [78]. These findings suggest that the unique keloid microenvironment or niche may preferentially recruit bone marrow MSCs and sustain their interaction with resident fibroblasts, thus contributing to keloid formation by producing abundant extracellular matrix at the scar site. The interaction between the keloid niche and keloid derived stem cells, KPCs, has been demonstrated here in our study, which is sustained by the IL-17/IL-6 feedback loop. It remains to be determined whether IL-17/IL-6 axis contributes to the recruitment of BMMSCs to keloid lesion and whether KPCs represent a distinct population of resident MSCs or derived from BMMSCs. In addition, a recent study reported that methylprednisolone is capable of inhibiting IL- 17 production in lymphocytes and lymph node cells [82]. Further studies are needed to determine whether the anti-scarring effect of the intralesional steroid therapy in keloid scar correlates with its role on the regulation of the IL- 17/IL-6. Using our established keloid-like animal models we will be able to further investigate the interplay between stem cells and their immediate niche.

In summary, we have successfully identified and characterized multipotent precursor cells from keloid scars and provide evidence supporting IL-17/IL-6 axis as an essential component of the unique keloid niche that provides extra proliferation-promoting signals to resident stem cells. Based on our findings, we believe that the inflammatory stimuli, i.e., inflammatory infiltrates and enriched cytokines, induced by wounding, surgical injury, or infection, may produce a persistent state of chronic inflammation at the wound site. This inflammatory niche of which IL-6 is a major component, will trigger recruitment and differentiation of other immune components, TH17 cells, which in conjunction with TGF61 stimulate further IL-6 secretion via IL- 17, creating an enriched proinflammatory cytokine milieu, and therefore, alter the functional niche. The altered niche can promote the resident stem cells to acquire a benign tumor-like stem cell phenotype characterized by increased cell proliferation and differentiation, therefore directly drives the benign tumor growth as seen in keloids (Figure 13). Findings from this study have not only substantiated our current knowledge regarding the role of adult stem cells in pathologic disease, specifically skin fibrosis, and other fibro-proliferative disorders, but most importantly, have provided a promising niche-related disease model, to further explore the intricate interactions between stem cells and their functional niche components, and ultimately lead to the development of an animal model for keloid fibrosis. Finally, this study should open a new avenue for stem cell research on benign tumor and lead to the rational design and development of innovative methods for prevention and treatment of niche-related diseases by specifically modulating or targeting the unique niche microenvironment of adult stem cells.

Many modifications and variations of the invention as hereinbefore set forth can be made without departing from the spirit and scope thereof and therefore only such limitations should be imposed as are indicated by the appended claims.

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

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Claims

What is claimed is:

1. Isolated keloid derived precursor cells (KPC), wherein the KPC exhibit stem cell properties.

2. The isolated KPC according to claim 1, wherein the stem cell properties comprise clonogenicity, multiple differentiation capacity, and self-renewal.

3. The isolated KPC according to claim 2, wherein the KPC are capable of multiple differentiation into mesoderm-derived adipocytes, osteoblasts, smooth muscle cell-like cells, or ectoderm- derived different types of neural cells.

4. A composition comprising isolated KPC according to claim 1.

5. A method of establishing a keloid animal model, comprising isolating KPC from keloid skin, transplanting them into an immunocompromised mouse, and allowing the cells to grow and form a keloid scar.

6. A method of treating keloid scarring comprising: a) obtaining a keloid animal model according to claim 5; b) administering an IL-6 neutralizing antibody into the scar of the model; and c) determining that a reduction in the size of the scar is indicative that the scar has been treated.

7. A method of preventing keloid scarring comprising: a) administering an IL-6 neutralizing antibody into an immunocompromised mouse; b) transplanting isolated KPC into the mouse and allowing them to grow and form a keloid scar; c) administering an IL-6 neutralizing antibody into the scar of the mouse; d) comparing the growth of the keloid scar in the mouse given the IL-6 neutralizing antibody with the growth of a keloid in a control mouse; and c) determining that the amount of keloid scarring in the mouse given the IL-6 neutralizing antibody is less than the amount of keloid scarring in the control mouse is indicative of the prevention of keloid scarring.

8. A method of treating keloid scarring, comprising decreasing IL-6 levels in a keloid scar by administering an IL-6 neutralizing antibody into the scar, and determining that a reduction in the size of the scar is indicative that the scar has been treated.

9. A method of preventing keloid scarring from occurring, comprising: a) administering IL-6 neutralizing antibody into a subject; b) administering KPC into the subject; c) comparing the growth of KPC in the subject given the IL-6 neutralizing antibody with the growth of KPC in a control subject; and d) determining that the amount of keloid scarring in the subject given the IL-6 neutralizing antibody is less than the amount of keloid scarring in the control subject is indicative of the prevention of keloid scarring.

10. A method of treating an inflammatory disease in a subject, comprising decreasing the IL-6 levels in the subject by administering an IL- 6 neutralizing antibody, and determining that a reduction in the amount of inflammation, is indicative of treatment of the inflammatory disease.

11. The method according to claim 10, wherein the inflammatory disease is chronic inflammatory or fibroproliferative.

12. The method according to claim 10, wherein the inflammatory disease is keloids, rheumatoid arthritis, inflammatory bowel disease (colitis), multiple sclerosis, benign tumors, or pulmonary fibrosis.

13. A method of screening for a pharmacological target of inflammatory diseases comprising contacting a pharmacological target with a IL-17/IL-6 inflammatory niche model and determining that changes in the amount of inflammation or an agent that causes inflammation, is indicative of the identification of a pharmacological target that may be used to treat an inflammatory disease.

14. The method of claim 13, wherein the inflammatory disease is chronic inflammatory or fibroproliferative.

15. The method according to claim 14, wherein the inflammatory disease is keloids, rheumatoid arthritis, inflammatory bowel disease (colitis), multiple sclerosis, benign tumors, or pulmonary fibrosis.