Hypoxic conditions promote a proliferative, poorly differentiated phenotype in COPD lung tissue progenitor cells in vitro.

PURPOSE: Chronic obstructive pulmonary disease (COPD) patients experience hypoxemia and lung tissue hypoxia, causing vasoconstriction, and at its most severe Cor pulmonale. However, minimal attention has been given to the effects of hypoxia at the cellular level. We hypothesize that a persistent progenitor cell population undergoes an aberrant differentiation process, influenced by changes in oxygen. METHODS: Distal lung progenitor cells from two emphysematous donors were cultured in 21% and 2% oxygen. Proliferation was determined on collagen-coated plastic and in 3T3-J2 co-culture. Epithelial (E-cadherin, pan-cytokeratin) and progenitor (TP63, cytokeratin 5) marker expressions were examined. Cells were differentiated at air-liquid interface, and ciliated, mucus-producing, and club cell populations identified by immunofluorescence. MUC5AC, MUC5B, CC10, and TP63 expression were determined using qRT-PCR, mucin5AC, and mucin5B protein levels by ELISA, and secreted mucin by periodic acid biotin hydrazide assay. RESULTS: Cells were positive for epithelial and progenitor markers at isolation and passage 5. Passage 5 cells in hypoxia increased the proportion of TP63 by 10% from 51.6 ± 1.2% to 62.6 ± 2.3% (p ≤ 0.01). Proliferative capacity was greater on 3T3J2 cells and in 2% oxygen, supporting the emergence of a proliferation unrestricted population with limited differentiation capacity. Differentiation resulted in ßIV tubulin positive-ciliated cells, mucin5AC, mucin5B, and CC10 positive secretory cells. Epithelial barrier formation was reduced (p ≤ 0.0001) in hypoxia-expanded cells. qRT-PCR showed higher mucin expression in 2% cells, significantly so with MUC5B (p ≤ 0.05). Although overall mucin5AC and mucin5B content was greater in 21% cells, normalization of secreted mucin to DNA showed a trend for increased mucin by low oxygen cells. CONCLUSIONS: These results demonstrate that hypoxia promotes a proliferative phenotype while affecting subsequent progenitor cell differentiation capacity. Furthermore, the retained differentiation potential becomes skewed to a more secretory phenotype, demonstrating that hypoxia may be contributing to disease symptoms and severity in COPD patients.

Chondrogenic Commitment of Human Bone Marrow Mesenchymal Stem Cells in a Perfused Collagen Hydrogel Functionalized with hTGF-ß1-Releasing PLGA Microcarrier.

Tissue engineering strategies can be relevant for cartilage repair and regeneration. A collagen matrix was functionalized with the addition of poly-lactic-co-glycolic acid microcarriers (PLGA-MCs) carrying a human Transforming Growth Factor ß1 (hTFG-ß1) payload, to provide a 3D biomimetic environment with the capacity to direct stem cell commitment towards a chondrogenic phenotype. PLGA-MCs (mean size 3 ± 0.9 µm) were prepared via supercritical emulsion extraction technology and tailored to sustain delivery of payload into the collagen hydrogel for 21 days. PLGA-MCs were coseeded with human Bone Marrow Mesenchymal Stem Cells (hBM-MSCs) in the collagen matrix. Chondrogenic induction was suggested when dynamic perfusion was applied as indicated by transcriptional upregulation of COL2A1 gene (5-fold; p < 0.01) and downregulation of COL1A1 (0.07-fold; p < 0.05) and COL3A1 (0.11-fold; p < 0.05) genes, at day 16, as monitored by qRT-PCR. Histological and quantitative-immunofluorescence (qIF) analysis confirmed cell activity by remodeling the synthetic extracellular matrix when cultured in perfused conditions. Static constructs lacked evidence of chondrogenic specific gene overexpression, which was probably due to a reduced mass exchange, as determined by 3D system Finite Element Modelling (FEM) analysis. Proinflammatory (IL-6, TNF, IL-12A, IL-1ß) and anti-inflammatory (IL-10, TGF-ß1) cytokine gene expression by hBM-MSC was observed only in dynamic culture (TNF and IL-1ß 10-fold, p < 0.001; TGF-ß1 4-fold, p < 0.01 at Day 16) confirming the cells' immunomodulatory activity mainly in relation to their commitment and not due to the synthetic environment. This study supports the use of 3D hydrogel scaffolds, equipped for growth factor controlled delivery, as tissue engineered models for the study of in vitro chondrogenic differentiation and opens clinical perspectives for injectable collagen-based advanced therapy systems.

Genetically engineered distal airway stem cell transplantation protects mice from pulmonary infection.

Severe pulmonary infection is a major threat to human health accompanied by substantial medical costs, prolonged inpatient requirements, and high mortality rates. New antimicrobial therapeutic strategies are urgently required to address the emergence of antibiotic resistance and persistent bacterial infections. In this study, we show that the constitutive expression of a native antimicrobial peptide LL-37 in transgenic mice aids in clearing Pseudomonas aeruginosa (PAO1), a major pathogen of clinical pulmonary infection. Orthotopic transplantation of adult mouse distal airway stem cells (DASCs), genetically engineered to express LL-37, into injured mouse lung foci enabled large-scale incorporation of cells and long-term release of the host defense peptide, protecting the mice from bacterial pneumonia and hypoxemia. Further, correlates of DASCs in adult humans were isolated, expanded, and genetically engineered to demonstrate successful construction of an anti-infective artificial lung. Together, our stem cell-based gene delivery therapeutic platform proposes a new strategy for addressing recurrent pulmonary infections with future translational opportunities.

Hypoxia-Modified Cancer Cell Metabolism.

While oxygen is critical to the continued existence of complex organisms, extreme levels of oxygen within a system, known as hypoxia (low levels of oxygen) and hyperoxia (excessive levels of oxygen), potentially promote stress within a defined biological environment. The consequences of tissue hypoxia, a result of a defective oxygen supply, vary in response to the gravity, extent and environment of the malfunction. Persistent pathological hypoxia is incompatible with normal biological functions, and as a result, multicellular organisms have been compelled to develop both organism-wide and cellular-level hypoxia solutions. Both direct, including oxidative phosphorylation down-regulation and inhibition of fatty-acid desaturation, and indirect processes, including altered hypoxia-sensitive transcription factor expression, facilitate the metabolic modifications that occur in response to hypoxia. Due to the dysfunctional vasculature associated with large areas of some cancers, sections of these tumors continue to develop in hypoxic environments. Crucial to drug development, a robust understanding of the significance of these metabolism changes will facilitate our understanding of cancer cell survival. This review defines our current knowledge base of several of the hypoxia-instigated modifications in cancer cell metabolism and exemplifies the correlation between metabolic change and its support of the hypoxic-adapted malignancy.

Tenogenic Differentiation of Human Embryonic Stem Cells.

Tendon healing is complex to manage because of the limited regeneration capacity of tendon tissue; stem cell-based tissue engineering approaches may provide alternative healing strategies. We sought to determine whether human embryonic stem cells (hESC) could be induced to differentiate into tendon-like cells by the addition of exogenous bone morphogenetic protein (BMP)12 (growth differentiation factor[GDF]7) and BMP13 (GDF6). hESC (SHEF-1) were maintained with or without BMP12/13 supplementation, or supplemented with BMP12/13 and the Smad signaling cascade blocking agent, dorsomorphin. Primary rat tenocytes were included as a positive control in immunocytochemistry analysis. A tenocyte-like elongated morphology was observed in hESC after 40-days continuous supplementation with BMP12/13 and ascorbic acid (AA). These cells displayed a tenomodulin expression pattern and morphology consistent with that of the primary tenocyte control. Analysis of tendon-linked gene transcription in BMP12/13 supplemented hESC demonstrated consistent expression of COL1A2, COL3A1, DCN, TNC, THBS4, and TNMD levels. Conversely, when hESCs were cultured in the presence of BMP12/13 and dorsomorphin COL3A1, DCN, and TNC gene expression and tendon matrix formation were inhibited. Taken together, we have demonstrated that hESCs are responsive to tenogenic induction via BMP12/13 in the presence of AA. The directed in vitro generation of tenocytes from pluripotent stem cells may facilitate the development of novel repair approaches for this difficult to heal tissue.

Pluripotent and Multipotent Stem Cells Display Distinct Hypoxic miRNA Expression Profiles.

MicroRNAs are reported to have a crucial role in the regulation of self-renewal and differentiation of stem cells. Hypoxia has been identified as a key biophysical element of the stem cell culture milieu however, the link between hypoxia and miRNA expression in stem cells remains poorly understood. We therefore explored miRNA expression in hypoxic human embryonic and mesenchymal stem cells (hESCs and hMSCs). A total of 50 and 76 miRNAs were differentially regulated by hypoxia (2% O2) in hESCs and hMSCs, respectively, with a negligible overlap of only three miRNAs. We found coordinate regulation of precursor and mature miRNAs under hypoxia suggesting their regulation mainly at transcriptional level. Hypoxia response elements were located upstream of 97% of upregulated hypoxia regulated miRNAs (HRMs) suggesting hypoxia-inducible-factor (HIF) driven transcription. HIF binding to the candidate cis-elements of specific miRNAs under hypoxia was confirmed by Chromatin immunoprecipitation coupled with qPCR. Role analysis of a subset of upregulated HRMs identified linkage to reported inhibition of differentiation while a downregulated subset of HRMs had a putative role in the promotion of differentiation. MiRNA-target prediction correlation with published hypoxic hESC and hMSC gene expression profiles revealed HRM target genes enriched in the cytokine:cytokine receptor, HIF signalling and pathways in cancer. Overall, our study reveals, novel and distinct hypoxia-driven miRNA signatures in hESCs and hMSCs with the potential for application in optimised culture and differentiation models for both therapeutic application and improved understanding of stem cell biology.

Immortalisation with hTERT Impacts on Sulphated Glycosaminoglycan Secretion and Immunophenotype in a Variable and Cell Specific Manner.

BACKGROUND: Limited options for the treatment of cartilage damage have driven the development of tissue engineered or cell therapy alternatives reliant on ex vivo cell expansion. The study of chondrogenesis in primary cells is difficult due to progressive cellular aging and senescence. Immortalisation via the reintroduction of the catalytic component of telomerase, hTERT, could allow repeated, longitudinal studies to be performed while bypassing senescent phenotypes. METHODS: Three human cell types: bone marrow-derived stromal cells (BMA13), embryonic stem cell-derived (1C6) and chondrocytes (OK3) were transduced with hTERT (BMA13H, 1C6H and OK3H) and proliferation, surface marker expression and tri-lineage differentiation capacity determined. The sulphated glycosaminoglycan (sGAG) content of the monolayer and spent media was quantified in maintenance media (MM) and pro-chondrogenic media (PChM) and normalised to DNA. RESULTS: hTERT expression was confirmed in transduced cells with proliferation enhancement in 1C6H and OK3H cells but not BMA13H. All cells were negative for leukocyte markers (CD19, CD34, CD45) and CD73 positive. CD14 was expressed at low levels on OK3 and OK3H and HLA-DR on BMA13 (84.8%). CD90 was high for BMA13 (84.9%) and OK3 (97.3%) and moderate for 1C6 (56.7%), expression was reduced in BMA13H (33.7%) and 1C6H (1.6%). CD105 levels varied (BMA13 87.7%, 1C6 8.2%, OK3 43.3%) and underwent reduction in OK3H (25.1%). 1C6 and BMA13 demonstrated osteogenic and adipogenic differentiation but mineralised matrix and lipid accumulation appeared reduced post hTERT transduction. Chondrogenic differentiation resulted in increased monolayer-associated sGAG in all primary cells and 1C6H (p<0.001), and BMA13H (p<0.05). In contrast OK3H demonstrated reduced monolayer-associated sGAG in PChM (p<0.001). Media-associated sGAG accounted for ≥55% (PChM-1C6) and ≥74% (MM-1C6H). CONCLUSION: In conclusion, hTERT transduction could, but did not always, prevent senescence and cell phenotype, including differentiation potential, was affected in a variable manner. As such, these cells are not a direct substitute for primary cells in cartilage regeneration research.

The application of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) scaffolds for tendon repair in the rat model.

Tendon injuries and defects present a substantial burden to global healthcare economies. There are no synthetic/biosynthesised implants available which can restore full function or match the mechanical properties of native tendon. Therefore, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) was investigated for its utility as a scaffold in a rat Achilles tendon repair model. Porous PHBHHx tubes and fibres were prepared with particle leaching and extrusion methods, respectively. Collagen gels reinforced by polymer fibres were inserted into the lumen of scaffold tubes to create the operational scaffold unit. Mechanical testing demonstrated that PHBHHx scaffolds had comparable mechanical properties to rat tendon, with maximal loads of 23.73 ± 1.08 N, compared to 17.35 ± 1.76 N in undamaged rat Achilles tendon. Sprague-Dawley (SD) rats were split into four experimental groups: control, PHBHHx scaffold only, PHBHHx scaffold and collagen, PHBHHx scaffold, collagen and tenocyte compositions for implantation to repair an induced Achilles tendon defect. No secondary immune response to PHBHHx was observed over a 40 days period of implantation. Movement was restored in PHBHHx scaffold-collagen-tenocyte recipient rats at an earlier time point than in other experimental groups, with complete load-bearing and function returning 20 days post-surgery as determined by the Achilles Functional Index. In vitro testing of tendon constructs after 40 days demonstrated reductions in PHBHHx molecular weight and polydispersity index accompanied by an increase in mean chain length indicating degradation of smaller polymer chain subunits. Similarly a reduction in PHBHHx tube ultimate tensile strength and elastic modulus was observed. Histological analysis provided evidence of tissue remodelling and cell alignment. In summary, PHBHHx scaffolds have been successfully applied in an in vivo tendon repair model raising promise for future utility in tissue engineering applications.