RESUMO
To explore how limbal niche cells (LNCs) may control quiescence, self-renewal, and corneal epithelial lineage commitment/differentiation of limbal epithelial progenitor/stem cells (LEPCs), we have established an in vitro sphere assay by reunion between the two cell types in three-dimensional Matrigel. The resultant sphere exhibits inhibition of corneal epithelial lineage commitment/differentiation and marked clonal growth of LEPCs, of which the latter is correlated with activation of canonical Wnt signaling. Herein, we have created a similar reunion assay in immobilized heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3), which is purified from amniotic membrane (AM) and consists of a complex formed by hyaluronic covalently linked to heavy chain 1 of inter-α-inhibitor and noncovalently linked to pentraxin 3. The resultant spheres exhibited similar suppression of corneal epithelial lineage commitment/differentiation but upregulation of quiescence markers including nuclear translocation of Bmi-1, and negligible clonal growth of LEPCs. This outcome was correlated with the suppression of canonical Wnt but activation of noncanonical (Planar cell polarity) Wnt signaling as well as BMP signaling in both LEPCs and LNCs. The activation of BMP signaling in LNCs was pivotal because nuclear translocation of pSmad1/5/8 was prohibited in hLEPCs when reunioned with mLNCs of conditionally deleted Bmpr1a;Acvr1(DCKO) mice. Furthermore, ablation of BMP signaling in LEPCs led to upregulation of cell cycle genes, downregulation of Bmi-1, nuclear exclusion of phosphorylated Bmi-1, and marked promotion of the clonal growth of LEPCs. Hence, HC-HA/PTX3 uniquely upregulates BMP signaling in LNCs which leads to BMP signaling in LEPCs to achieve quiescence, helping explain how AM transplantation is clinically useful to be used as a matrix for ex vivo expansion of LEPCs and to treat corneal blindness caused by limbal stem cells deficiency.
Assuntos
Âmnio/metabolismo , Proteínas Morfogenéticas Ósseas/biossíntese , Proteína C-Reativa/biossíntese , Células Epiteliais/metabolismo , Ácido Hialurônico/biossíntese , Componente Amiloide P Sérico/biossíntese , Nicho de Células-Tronco/fisiologia , Células 3T3 , Animais , Proteína C-Reativa/isolamento & purificação , Diferenciação Celular/fisiologia , Células Cultivadas , Humanos , Ácido Hialurônico/isolamento & purificação , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/isolamento & purificação , Camundongos , Camundongos Transgênicos , Componente Amiloide P Sérico/isolamento & purificação , Transdução de Sinais/fisiologia , Células-Tronco/metabolismoRESUMO
Pain after surgery causes significant suffering. Opioid analgesics cause severe side effects and accidental death. Therefore, there is an urgent need to develop non-opioid therapies for managing post-surgical pain. Local application of Clarix Flo (FLO), a human amniotic membrane (AM) product, attenuated established post-surgical pain hypersensitivity without exhibiting known side effects of opioid use in mice. This effect was achieved through direct inhibition of nociceptive dorsal root ganglion (DRG) neurons via CD44-dependent pathways. We further purified the major matrix component, the heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) from human AM that has greater purity and water solubility than FLO. HC-HA/PTX3 replicated FLO-induced neuronal and pain inhibition. Mechanistically, HC-HA/PTX3 induced cytoskeleton rearrangements to inhibit sodium current and high-voltage activated calcium current on nociceptive neurons, suggesting it is a key bioactive component mediating pain relief. Collectively, our findings highlight the potential of naturally derived biologics from human birth tissues as an effective non-opioid treatment for post-surgical pain and unravel the underlying mechanisms.
RESUMO
Gram-positive rod-shaped thermophilic bacteria were isolated using samples collected from terrestrial natural thermal spring located at Unkeshwar (Longitude 78.22 degree East to 78.34 degree East, Latitude 19 degree 34' North to 19 degree 40' North), District Nanded, Maharashtra State, India. The isolates were then cultivated using selective media and identified using culture-dependent techniques. One prominent isolate (UN1) exhibited high temperature stability and remarkable amylase production and was identified as Bacillus licheniformis. Amylase production was carried out in starch media and the enzyme was partially purified and characterized for optimization of pH and temperature. Amylolytic activity of the enzyme was determined. Nanoactivator-mediated modifications were carried out to enhance amylolytic activity of the partially purified amylase. Three-fold increase in catalytic efficiency of amylase was obtained after modification.
Assuntos
Amilases/metabolismo , Bacillus/enzimologia , Cálcio/química , Nanoestruturas , Fosfatos/química , Amilases/química , Bacillus/isolamento & purificação , Catálise , Temperatura Alta , Concentração de Íons de Hidrogênio , NanotecnologiaRESUMO
In the developing area of modern nanobiotechnology, the research is being focused on enhancement of catalytic performance in terms of efficiency and stability of enzymes to fulfill the industrial demand. In the context of this interdisciplinary era, we isolated and identified alkaline protease producer Bacillus aryabhattai P1 by polyphasic approach and then followed one variable at a time approach to optimize protease production from P1. The modified components of fermentation medium (g/L) were wheat bran 10, soybean flour 10, yeast extract 5, NaCl 10, KH2PO4 1, K2HPO4 1 and MgSO4·7H2O 0.2 (pH 9). The optimum alkaline protease production from P1 was recorded 75 ± 3 U/mg at 35 °C and pH 9 after 96 h of fermentation period. Molecular weight of partially purified P1 alkaline protease was 26 KDa as revealed by SDS-PAGE. Calcium based nanoceramic material was prepared by wet chemical precipitation method and doped in native P1 protease for catalytic activity enhancement. Catalytic activity of modified P1 protease was attained by nanoactivator mediated modulation was more by 5.58 fold at pH 10 and 30 °C temperature. The nanoceramic material named as nanoactivator, with grain size of 40-60 nm was suitable to redesign the active site of P1 protease. Such types of modified proteases can be used in different nanobiotechnological applications.
RESUMO
On ocular surface, corneal epithelial stem cells (SC) reside in limbus between cornea and conjunctiva. Pax6, an evolutionally conserved transcription factor essential for eye development, is expressed in post-natal corneal and limbal epithelia progenitors (LEPC) but not in underlying stroma. Because Pax6 is transiently expressed in developing corneal stroma and a subset of limbal and corneal stromal progenitors, we examined the role of Pax6 in limbal niche cells (LNC) in maintaining the phenotype of neural crest (NC) progenitors to support LEPC. Our results showed that nuclear Pax6 staining was found in freshly isolated LNC but not corneal stromal cells. Serial passaged LNC resulted in gradual loss of nuclear Pax6 (46 kDa) staining and neural crest progenitor status defined by the expression of embryonic SCs and NC markers, neurosphere formation, and differentiation into neurons, oligodendrocytes and astrocytes. Gain of function of 46 kDa Pax6 in late-passaged LNC resulted in nuclear Pax6 staining and promotion of the aforementioned NC progenitor status. In an in vitro reunion assay, early passaged LNC and late passaged LNC with overexpression of Pax6 inhibited the expression of corneal epithelial differentiation marker and promoted holoclone by LEPC. Therefore, expression of nuclear 46 kDa Pax6 in LNC plays an important developmental role in maintaining NC progenitor status to support self-renewal of corneal epithelial SCs in the limbal niche.
Assuntos
Autorrenovação Celular/genética , Epitélio Corneano/citologia , Limbo da Córnea/citologia , Crista Neural/citologia , Crista Neural/metabolismo , Fator de Transcrição PAX6/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Biomarcadores , Diferenciação Celular/genética , Células Cultivadas , Expressão Gênica , Genes Reporter , HumanosRESUMO
Proliferative vitreoretinopathy (PVR) is mediated by proliferation and epithelial mesenchymal transition (EMT) of retinal pigment epithelium (RPE). Because heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) purified from human amniotic membrane exerts anti-inflammatory and anti-scarring actions, we hypothesized that HC-HA/PTX3 could inhibit these PVR-related processes in vitro. In this study, we first optimized an ARPE-19 cell culture model to mimic PVR by defining cell density, growth factors, and cultivation time. Using this low cell density culture model and HA as a control, we tested effects of HC-HA/PTX3 on the cell viability (cytotoxicity), proliferation (EGF + FGF-2) and EMT (TGF-ß1). Furthermore, we determined effects of HC-HA/PTX3 on cell migration (EGF + FGF-2 + TGF-ß1) and collagen gel contraction (TGF-ß1). We found both HA and HC-HA/PTX3 were not toxic to unstimulated RPE cells. Only HC-HA/PTX3 dose-dependently inhibited proliferation and EMT of stimulated RPE cells by down-regulating Wnt (ß-catenin, LEF1) and TGF-ß (Smad2/3, collagen type I, α-SMA) signaling, respectively. Additionally, HA and HC-HA/PTX3 inhibited migration but only HC-HA/PTX3 inhibited collagen gel contraction. These results suggest HC-HA/PTX3 is a non-toxic, potent inhibitor of proliferation and EMT of RPE in vitro, and HC-HA/PTX3's ability to inhibit PVR formation warrants evaluation in an animal model.
Assuntos
Proteína C-Reativa/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Ácido Hialurônico/farmacologia , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Componente Amiloide P Sérico/farmacologia , Biomarcadores , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/patologia , Humanos , Transporte Proteico , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Vitreorretinopatia Proliferativa/etiologia , Vitreorretinopatia Proliferativa/metabolismo , Vitreorretinopatia Proliferativa/patologiaRESUMO
Chronic graft-versus-host disease (cGVHD) is a major complication of hematopoietic stem cell transplantation. Dry eye disease is the prominent ocular sequel of cGVHD and is caused by excessive inflammation and fibrosis in the lacrimal glands. Heavy chain-Hyaluronan/Pentraxin 3 (HC-HA/PTX3) is a complex purified from human amniotic membrane (AM) and known to exert anti-inflammatory and anti-scarring actions. In this study, we utilized a mouse model of cGVHD to examine whether HC-HA/PTX3 could attenuate dry eye disease elicited by cGVHD. Our results indicated that subconjunctival and subcutaneous injection of HC-HA/PTX3 preserved tear secretion and conjunctival goblet cell density and mitigated inflammation and scarring of the conjunctiva. Such therapeutic benefits were associated with suppression of scarring and infiltration of inflammatory/immune cells in the lacrimal glands. Furthermore, HC-HA/PTX3 significantly reduced the extent of infiltration of CD45+ CD4+ IL-17+ cells, CD45+ CD34+ collagen I+ CXCR4+ fibrocytes, and HSP47+ activated fibroblasts that were accompanied by upregulation of collagen type Iα1, collagen type IIIα1 and NF-kB in lacrimal glands. Collectively, these pre-clinical data help prove the concept that subcutaneous and subconjunctival injection of HC-HA/PTX3 is a novel approach to prevent dry eye disease caused by cGVHD and allow us to test its safety and efficacy in future human clinical trials.
Assuntos
Proteína C-Reativa/farmacologia , Cicatriz/prevenção & controle , Túnica Conjuntiva/efeitos dos fármacos , Síndromes do Olho Seco/tratamento farmacológico , Doença Enxerto-Hospedeiro/patologia , Ácido Hialurônico/farmacologia , Aparelho Lacrimal/efeitos dos fármacos , Componente Amiloide P Sérico/farmacologia , Âmnio/química , Animais , Transplante de Medula Óssea/efeitos adversos , Proteína C-Reativa/isolamento & purificação , Doença Crônica , Cicatriz/etiologia , Cicatriz/imunologia , Cicatriz/patologia , Túnica Conjuntiva/imunologia , Túnica Conjuntiva/patologia , Modelos Animais de Doenças , Síndromes do Olho Seco/etiologia , Síndromes do Olho Seco/imunologia , Síndromes do Olho Seco/patologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Fibroblastos/patologia , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/imunologia , Humanos , Ácido Hialurônico/isolamento & purificação , Injeções Intraoculares , Injeções Subcutâneas , Aparelho Lacrimal/imunologia , Aparelho Lacrimal/patologia , Masculino , Camundongos , Componente Amiloide P Sérico/isolamento & purificação , Transplante Homólogo , Irradiação Corporal TotalRESUMO
PURPOSE: Adipose-derived stem cells (ASCs) have gained importance due to their myriad potential clinical applications. We hypothesize that progenitor cells also exist besides those conventionally isolated from the stromal vascular fraction (SVF). METHOD: Central and medial orbital adipose tissues obtained from patients during eyelid surgery were digested with collagenase for 3 or 16 hours at 37°C with or without shaking. After centrifugation, the remaining cell pellet was resuspended and filtered to yield flow through in SVF and retained cells (RC) on the filter. Single cells from RC and SVF were cultured on 5% coated Matrigel in serum-free modified embryonic stem cells medium (MESCM) for 10 passages. The progenitor status was evaluated by the expression of a number of markers by qPCR and immunofluorescence staining as well as their plasticity for endothelial and tri-lineage differentiation. RESULTS: Type I collagenase digestion for 3 hours under shaking was significantly less effective in releasing progenitor cells than collagenase A digestion for 16 hours without shaking. Following filtration, cells in SVF and RC, of which the latter were tangled in collagen IV-containing matrix, expressed different markers of progenitor cells. Cells from SVF and RC could be expanded for 10 passages on coated Matrigel in MESCM and exhibited similar or better potential to differentiate into vascular endothelial cells, chondrocytes, osteocytes, and adipocytes than SVF cells expanded on plastic in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). CONCLUSIONS: Different progenitor cells can be isolated and expanded from orbital adipose tissues. Further characterization of their mesodermal or neuroectodermal origin might enhance clinical outcome when used as a source of autologous stem cells for ocular surface regeneration.
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Tecido Adiposo/citologia , Células-Tronco Mesenquimais/citologia , Adipócitos/citologia , Adipogenia , Idoso , Idoso de 80 Anos ou mais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , ÓrbitaRESUMO
PURPOSE: Transplantation of ex vivo expanded limbal epithelial progenitor cells (LEPCs) on epithelially denuded amniotic membrane (dAM) in supplemented hormonal epithelial medium (SHEM) is an alternative solution for treating corneal blindness due to limbal stem cell (SC) deficiency. Because the phenotype of limbal niche cells (NCs) is preserved better in serum-free modified embryonic stem cell (ESC) medium (MESCM) than SHEM, we question whether the aforementioned expansion protocol can be further optimized by maintaining limbal NCs using MESCM. METHODS: Collagenase-isolated limbal clusters were cultured on dAM in SHEM or MESCM for 8 to 10 days. Epithelial outgrowth sheets removed by dispase were subjected to real-time quantitative polymerase chain reaction (qPCR) and immunostaining for expression of corneal epithelial markers (p63α, pax6, and K12) and NC markers (FLK-1, CD34, CD31, PDGFR-B, and α-SMA). A total of 1000 single cells were seeded on 6-well dish containing 3T3 feeder layers for 12 to 14 days before rhodamine B staining. RESULTS: Epithelial outgrowth in SHEM showed a significant loss of corneal SC and ESC markers when compared with freshly collagenase-isolated limbal clusters. Although the epithelial outgrowth was slower in MESCM, epithelial cell size was consistently smaller than that found in SHEM. Furthermore, MESCM maintained a significantly higher percentage of PCK-/ Vim+ cells and exhibited a significant upregulation of NC markers and corneal epithelial SC markers (K15, Bmi-1, and Msi-1) than SHEM. Furthermore, the number of purported holoclones was significantly promoted in MESCM than SHEM. CONCLUSION: These data collectively suggest that MESCM can be used to replace SHEM to further promote expansion of LEPC by maintaining limbal native NCs. TRANSLATIONAL RELEVANCE: Effective ex vivo expansion of limbal epithelial SC is a first and important step toward the success of treating corneal blindness caused by limbal stem cell deficiency and paves the way for future applications in regenerative medicine.