Understanding cornea epithelial stem cells and stem cell deficiency: Lessons learned using vertebrate model systems

Animal models have contributed greatly to our understanding of human diseases. Here, we focus on cornea epithelial stem cell (CESC) deficiency (commonly called limbal stem cell deficiency, LSCD). Corneal development, homeostasis and wound healing are supported by specific stem cells, that include the CESCs. Damage to or loss of these cells results in blindness and other debilitating ocular conditions. Here we describe the contributions from several vertebrate models toward understanding CESCs and LSCD treatments. These include both mammalian models, as well as two aquatic models, Zebrafish and the amphibian, Xenopus. Pioneering developments have been made using stem cell transplants to restore normal vision in patients with LSCD, but questions still remain about the basic biology of CESCs, including their precise cell lineages and behavior in the cornea. We describe various cell lineage tracing studies to follow their patterns of division, and the fates of their progeny during development, homeostasis, and wound healing. In addition, we present some preliminary results using the Xenopus model system. Ultimately, a more thorough understanding of these cornea cells will advance our knowledge of stem cell biology and lead to better cornea disease therapeutics.     

Time Kinetics of Hemoglobin and Myoglobin Activation by Tetrachlorodecaoxide (TCDO)

In the presence of peroxidase, myoglobin or hemoglobin, Tetrachlorodecaoxide (TCDO) forms an active oxygen species which is similar to the product of the polymorphonuclear leucocyte (PMNL) myeloperoxidase reaction and the “Klebanoff Model” of phagocytosis, but it is also produced under anaerobic conditions. Randomly destructive species such as the free OH^- radical or singlet oxygen are not formed. The kinetics of the heme-dependent activation vary according to the heme type present. In comparison to myoglobin, blood shows a 2 h delay in the appearance of maximal activity. On the basis of known biochemical and clinical-physiological data, a hypothesis can be proposed to explain the reoxygenation observed in hypoxic tissue, induced by TCDO via this activated heme species. Under normal physiological conditions, vasodilation occurs via catalysis by xanthine oxidase or PMNL-dependent activation of fatty acids.     

Oncogenes and inflammation rewire host energy metabolism in the tumor microenvironment

Here, we developed a model system to evaluate the metabolic effects of oncogene(s) on the host microenvironment. A matched set of “normal” and oncogenically transformed epithelial cell lines were co-cultured with human fibroblasts, to determine the “bystander” effects of oncogenes on stromal cells. ROS production and glucose uptake were measured by FACS analysis. In addition, expression of a panel of metabolic protein biomarkers (Caveolin-1, MCT1, and MCT4) was analyzed in parallel. Interestingly, oncogene activation in cancer cells was sufficient to induce the metabolic reprogramming of cancer-associated fibroblasts toward glycolysis, via oxidative stress. Evidence for “metabolic symbiosis” between oxidative cancer cells and glycolytic fibroblasts was provided by MCT1/4 immunostaining. As such, oncogenes drive the establishment of a stromal-epithelial “lactate-shuttle”, to fuel the anabolic growth of cancer cells. Similar results were obtained with two divergent oncogenes (RAS and NFκB), indicating that ROS production and inflammation metabolically converge on the tumor stroma, driving glycolysis and upregulation of MCT4. These findings make stromal MCT4 an attractive target for new drug discovery, as MCT4 is a shared endpoint for the metabolic effects of many oncogenic stimuli. Thus, diverse oncogenes stimulate a common metabolic response in the tumor stroma. Conversely, we also show that fibroblasts protect cancer cells against oncogenic stress and senescence by reducing ROS production in tumor cells. Ras-transformed cells were also able to metabolically reprogram normal adjacent epithelia, indicating that cancer cells can use either fibroblasts or epithelial cells as “partners” for metabolic symbiosis. The antioxidant N-acetyl-cysteine (NAC) selectively halted mitochondrial biogenesis in Ras-transformed cells, but not in normal epithelia. NAC also blocked stromal induction of MCT4, indicating that NAC effectively functions as an “MCT4 inhibitor”. Taken together, our data provide new strategies for achieving more effective anticancer therapy. We conclude that oncogenes enable cancer cells to behave as selfish “metabolic parasites”, like foreign organisms (bacteria, fungi, viruses). Thus, we should consider treating cancer like an infectious disease, with new classes of metabolically targeted “antibiotics” to selectively starve cancer cells. Our results provide new support for the “seed and soil” hypothesis, which was first proposed in 1889 by the English surgeon, Stephen Paget.     

Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins,Types, and Interactions

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that participate in regenerating tissue, tumor formation, or vasculogenesis, we devised a multi-colored cellular transplant model of tumor development in which cell populations originate from different fluorescently colored reporter gene mice and are transplanted, engrafted or injected in and around a developing tumor. These colored cells are then recruited and incorporated into the tumor stroma. In order to quantitatively assess bone marrow derived tumor stromal cells, we transplanted GFP expressing transgenic whole bone marrow into lethally irradiated RFP expressing mice as approved by IACUC. 0ovarian tumors that were orthotopically injected into the transplanted mice were excised 6-8 weeks post engraftment and analyzed for bone marrow marker of origin (GFP) as well as antibody markers to detect tumor associated stroma using multispectral imaging techniques. We then adapted a methodology we call MIMicc- Multispectral Interrogation of Multiplexed cellular compositions, using multispectral unmixing of fluoroprobes to quantitatively assess which labeled cell came from which starting populations (based on original reporter gene labels), and as our ability to unmix 4, 5, 6 or more spectra per slide increases, we''ve added additional immunohistochemistry associated with cell lineages or differentiation to increase precision. Utilizing software to detect co-localized multiplexed-fluorescent signals, tumor stromal populations can be traced, enumerated and characterized based on marker staining.¹     

Palifermin for the protection and regeneration of epithelial tissues following injury: new findings in basic research and pre‐clinical models

Keratinocyte growth factor (KGF) is a paracrine‐acting epithelial mitogen produced by cells of mesenchymal origin, that plays an important role in protecting and repairing epithelial tissues. Pre‐clinical data initially demonstrated that a recombinant truncated KGF (palifermin) could reduce gastrointestinal injury and mortality resulting from a variety of toxic exposures. Furthermore, the use of palifermin in patients with hematological malignancies reduced the incidence and duration of severe oral mucositis experienced after intensive chemoradiotherapy. Based upon these findings, as well as the observation that KGF receptors are expressed in many, if not all, epithelial tissues, pre‐clinical studies have been conducted to determine the efficacy of palifermin in protecting different epithelial tissues from toxic injury in an attempt to model various clinical situations in which it might prove to be of benefit in limiting tissue damage. In this article, we review these studies to provide the pre‐clinical background for clinical trials that are described in the accompanying article and the rationale for additional clinical applications of palifermin.     

Dental follicle progenitor cells responses to Porphyromonas gingivalis LPS

Periodontitis is a bacterially induced chronic inflammatory disease. Dental follicle progenitor cells (DFPCs) have been proposed as biological graft for periodontal regenerative therapies. The potential impact of bacterial toxins on DFPCs properties is still poorly understood. The aim of this study was to investigate whether DFPCs are able to sense and respond to lipopolysaccharide (LPS) from Porphyromonas gingivalis, a major periopathogenic bacterium. Specifically, we hypothesized that LPS could influence the migratory capacity and IL‐6 secretion of DFPCs. DFPCs properties were compared to bone marrow mesenchymal stem cells (BMSCs), a well‐studied class of adult stem cells. The analysis by flow cytometry indicated that DFPCs, similar to BMSCs, expressed low levels of both toll‐like receptor (TLR) 2 and 4. The TLR4 mRNA expression was down‐regulated in response to LPS in both cell populations, while on protein level TLR4 was significantly up‐regulated on BMSCs. The TLR2 expression was not influenced by the LPS treatment in both DFPCs and BMSCs. The migratory efficacy of LPS‐treated DFPCs was evaluated by in vitro scratch wound assays and found to be significantly increased. Furthermore, we assayed the secretion of interleukin‐6 (IL‐6), a potent stimulator of cell migration. Interestingly, the levels of IL‐6 secretion of DFPCs and BMSCs remained unchanged after the LPS treatment. Taken together, these results suggest that DFPCs are able to sense and respond to P. gingivalis LPS. Our study provides new insights into understanding the physiological role of dental‐derived progenitor cells in sites of periodontal infection.     

An in situ collagen-HA hydrogel system promotes survival and preserves the proangiogenic secretion of hiPSC-derived vascular smooth muscle cells

Human-induced pluripotent stem cell-derived vascular smooth muscle cells (hiPSC-VSMCs) with proangiogenic properties have huge therapeutic potential. While hiPSC-VSMCs have already been utilized for wound healing using a biomimetic collagen scaffold, an in situ forming hydrogel mimicking the native environment of skin offers the promise of hiPSC-VSMC mediated repair and regeneration. Herein, the impact of a collagen type-I-hyaluronic acid (HA) in situ hydrogel cross-linked using a polyethylene glycol-based cross-linker on hiPSC-VSMCs viability and proangiogenic paracrine secretion was investigated. Our study demonstrated increases in cell viability, maintenance of phenotype and proangiogenic growth factor secretion, and proangiogenic activity in response to the conditioned medium. The optimally cross-linked and functionalized collagen type-I/HA hydrogel system developed in this study shows promise as an in situ hiPSC-VSMC carrier system for wound regeneration.     

A possible role for inducible arginase isoform (AI) in the pathogenesis of chronic venous leg ulcer

Chronic venous ulcer (CVU) is a major cause of chronic wounds of lower extremities and presents a significant financial and resource burden to health care systems worldwide. Defects in the vasculature, matrix deposition, and re-epithelialization are the main histopathological changes believed to impede healing. Supplementation of the amino acid arginine that plays a crucial role in the interactions that occur during inflammation and wound healing was proven clinically to improve acute wound healing probably through enhancing activity of inducible arginase (AI) locally in the wounds. However, the possible mechanism of arginine action and the potential beneficial effects of AI/arginine in human chronic wounds remain unclear. In the present study, using biopsies, taken under local anesthesia, from adult patients (n = 12, mean age 55 years old) with CVUs in lower extremities, we investigated the correlation between AI distribution in CVUs and the histopathological changes, mainly proliferative and vascular changes. Our results show a distinct spatial distribution of AI along the ulcer in the epidermis and in the dermis with the highest level of expression being at the ulcer edge and the least expression towards the ulcer base. The AI cellular immunoreactivity, enzymatic activity, and protein levels were significantly increased towards the ulcer edge. Interestingly, a similar pattern of expression was encountered in the proliferative and the vascular changes with strong correlations between AI and the proliferative activity and vascular changes. Furthermore, AI cellular distribution was associated with increased proliferative activity, inflammation, and vascular changes. Our findings of differential expression of AI along the CVU base, edge, and nearby surrounding skin and its associations with increased proliferative activity and vascular changes provide further support to the AI implication in CVU pathogenesis. The presence of high levels of AI in the epidermis of chronic wounds may serve as a molecular marker of impaired healing and may provide future targets for therapeutic intervention.