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Dry eye disease (DED) is a multifactorial aging disorder leading to tear film insufficiency and instability. Yet, an important knowledge gap lingers in understanding senescence-associated ocular pathogenesis, due to limited in vitro translational lacrimal gland (LG) models. Consequently, this remains a major roadblock to discover effective therapies for the restoration of tear film secretion. Herein, the authors reported the magnetic bioassembly of two LG organoid platforms to recapitulate functional and aging states. Using a proof-of-concept approach, porcine primary LG cells were assembled into organoids via a magnetic 3D bioprinting (M3DB) platform. This platform could form reproducible LG organoids with epithelial hallmarks (AQP5+) and exhibit epithelial secretory functions (lysozyme activity). DNA damage-induced senescence and cell death was induced with etoposide, and LG organoid hypofunction and senescence-associated pathogenesis were observed. To confer DNA protection against aging, a novel gene therapy with Box A domain of high-mobility group box-1 (HMGB1-Box A) previously established by our group, was applied here to prevent LG cellular senescence for the first time. HMGB1-Box A transfection prevented LG organoids from senescence-associated pathogenesis at the transcriptomic, metabolomic and proteomic levels. Thus, M3DB platforms could generate functional and DNA damage-induced senescence LG organoids, and this latter damage could be prevented with HMGB1-Box A gene therapy.
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Senescencia Celular , Terapia Genética , Proteína HMGB1 , Aparato Lagrimal , Organoides , Organoides/metabolismo , Animales , Proteína HMGB1/metabolismo , Proteína HMGB1/genética , Porcinos , Terapia Genética/métodos , Aparato Lagrimal/metabolismo , Aparato Lagrimal/patología , Síndromes de Ojo Seco/terapia , Síndromes de Ojo Seco/metabolismo , Síndromes de Ojo Seco/patología , Humanos , Daño del ADNRESUMEN
BACKGROUND: Mesenchymal stromal/stem cells (MSCs) play a critical role in wound healing. Corlicyte® is an MSC product derived from allogeneic umbilical cord tissue donated under an institutional review board-approved protocol and processed in accordance with section 501(a)(2)(B) of the Federal Food, Drug, and Cosmetic Act. This open-label phase 1 trial was performed under a United States Food and Drug Administration Investigational New Drug Application to establish the safety and tolerability of Corlicyte® in patients with diabetes and chronic diabetic foot ulcer (DFU). METHODS: Escalating doses were applied topically twice a week for up to 8 weeks after ulcer debridement, wound photography, and measurement. Subjects were followed for 4 weeks after the treatment phase. Adverse events were assessed at every visit. RESULTS: Nine subjects in 2 dosing cohorts completed the trial. No subjects experienced a serious adverse reaction to Corlicyte® or the development of anti-human leukocyte antigen (HLA) antibodies. Sixty percentage of subjects in the lower dose cohort experienced ulcer closure by Day 70 of follow-up, while the mean ulcer size was reduced by 54-67% in the other subjects. CONCLUSIONS: Topical administration of Corlicyte®, a novel biologic therapy consisting of allogeneic umbilical cord lining MSCs, appeared safe and tolerable and resulted in a significant decrease in ulcer area, demonstrating its potential as a therapy for healing of chronic DFU.
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Hyposalivation and severe dry mouth syndrome are the most common complications in patients with head and neck cancer (HNC) after receiving radiation therapy. Conventional treatment for hyposalivation relies on the use of sialogogues such as pilocarpine; however, their efficacy is constrained by the limited number of remnant acinar cells after radiation. After radiotherapy, the salivary gland (SG) secretory parenchyma is largely destroyed, and due to the reduced stem cell niche, this gland has poor regenerative potential. To tackle this, researchers must be able to generate highly complex cellularized 3D constructs for clinical transplantation via technologies, including those that involve bioprinting of cells and biomaterials. A potential stem cell source with promising clinical outcomes to reserve dry mouth is adipose mesenchymal stem cells (AdMSC). MSC-like cells like human dental pulp stem cells (hDPSC) have been tested in novel magnetic bioprinting platforms using nanoparticles that can bind cell membranes by electrostatic interaction, as well as their paracrine signals arising from extracellular vesicles. Both magnetized cells and their secretome cues were found to increase epithelial and neuronal growth of in vitro and ex vivo irradiated SG models. Interestingly, these magnetic bioprinting platforms can be applied as a high-throughput drug screening system due to the consistency in structure and functions of their organoids. Recently, exogenous decellularized porcine ECM was added to this magnetic platform to stimulate an ideal environment for cell tethering, proliferation, and/or differentiation. The combination of these SG tissue biofabrication strategies will promptly allow for in vitro organoid formation and establishment of cellular senescent organoids for aging models, but challenges remain in terms of epithelial polarization and lumen formation for unidirectional fluid flow. Current magnetic bioprinting nanotechnologies can provide promising functional and aging features to in vitro craniofacial exocrine gland organoids, which can be utilized for novel drug discovery and/or clinical transplantation.
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Bioimpresión , Xerostomía , Humanos , Animales , Porcinos , Glándulas Salivales , Células Madre , RegeneraciónRESUMEN
Epidermal growth factor (EGF) is a known signaling cue essential towards the development and organoid biofabrication particularly for exocrine glands. This study developed an in vitro EGF delivery platform with Nicotiana benthamiana plant-produced EGF (P-EGF) encapsulated on hyaluronic acid/alginate (HA/Alg) hydrogel to improve the effectiveness of glandular organoid biofabrication in short-term culture systems. Primary submandibular gland epithelial cells were treated with 5 - 20 ng/mL of P-EGF and commercially available bacteria-derived EGF (B-EGF). Cell proliferation and metabolic activity were measured by MTT and luciferase-based ATP assays. P-EGF and B-EGF 5 - 20 ng/mL promoted glandular epithelial cell proliferation during 6 culture days on a comparable fashion. Organoid forming efficiency and cellular viability, ATP-dependent activity and expansion were evaluated using two EGF delivery systems, HA/Alg-based encapsulation and media supplementation. Phosphate buffered saline (PBS) was used as a control vehicle. Epithelial organoids fabricated from PBS-, B-EGF-, and P-EGF-encapsulated hydrogels were characterized genotypically, phenotypically and by functional assays. P-EGF-encapsulated hydrogel enhanced organoid formation efficiency and cellular viability and metabolism relative to P-EGF supplementation. At culture day 3, epithelial organoids developed from P-EGF-encapsulated HA/Alg platform contained functional cell clusters expressing specific glandular epithelial markers such as exocrine pro-acinar (AQP5, NKCC1, CHRM1, CHRM3, Mist1), ductal (K18, Krt19), and myoepithelial (α-SMA, Acta2), and possessed a high mitotic activity (38-62% Ki67 cells) with a large epithelial progenitor population (â¼70% K14 cells). The P-EGF encapsulation strikingly upregulated the expression of pro-acinar AQP5 cells through culture time when compared to others (B-EGF, PBS). Thus, the utilization of Nicotiana benthamiana in molecular farming can produce EGF biologicals amenable to encapsulation in HA/Alg-based in vitro platforms, which can effectively and promptly induce the biofabrication of exocrine gland organoids.
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Factor de Crecimiento Epidérmico , Hidrogeles , Factor de Crecimiento Epidérmico/farmacología , Agricultura Molecular , Organoides , Ácido Hialurónico/farmacología , Adenosina TrifosfatoRESUMEN
BACKGROUND: Significant improvement in skin tone was reported after topical application of a facial cream (CALECIM® Professional Multi-Action Cream, CALECIM Cosmeceuticals, Singapore) containing conditioned media (CM) derived from Red Deer Umbilical Cord Lining Mesenchymal Stem Cell (RD-CLMSC) culture. This study investigates the paracrine effects of RD-CLMSC-CM on human dermal fibroblasts (HDF) to understand how it may increase skin turgor and elasticity. Skin aging is associated with lower levels of extracellular matrix components such as hyaluronic acid (HA) and elastin, resulting in poor skin turgor and elasticity. Histochemical staining followed by photocolorimetry demonstrated that RD-CLMSC-CM upregulated HDF expression of elastin by 56% and HA by 83% compared with DMEM/10% Fetal Calf Serum (FCS).To further quantify the effects of CM, a proliferation assay was used to assess HDF response to RD-CLMSC-CM exposure. Exposure to RD-CLMSC-CM resulted in the highest increase in HDF proliferation over DMEM/10% FCS (113%) followed by Human (H)-CLMSC-CM (112%), then Human Foreskin Fibroblast (FSF)-CM (16%).These experimental results demonstrate both the cross-species efficacy and lack of toxicity of RD-CLMSC-CM on HDF. These pre-clinical studies also suggest the clinical effects of RD-CLMSC-CM on skin turgor may be related to increased HA and elastin production by HDF, as well as enhanced proliferation. J Drugs Dermatol. 2023;21(1):82-89. doi:10.36849/JDD.6906.
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Ciervos , Células Madre Mesenquimatosas , Humanos , Animales , Medios de Cultivo Condicionados , Elastina/metabolismo , Ciervos/metabolismo , Células Madre Mesenquimatosas/metabolismo , Cordón Umbilical , Fibroblastos/metabolismoRESUMEN
In Vietnam, helminthioses remain a major threat to public health and contribute to the maintenance of poverty in highly endemic regions. Through increased awareness of the damaging effects caused by helminthioses, the Vietnamese government has implemented many national programs over the past 30 years for the prevention and control of the most important helminthioses, such as, lymphatic filariasis, soil transmitted helminths, food borne zoonotic helminths, and others. Various control strategies have been applied to reduce or eliminate these worms, e.g. mass drug administration, economic development, control of vectors or intermediate hosts, public health interventions through education, proper composting procedures for excreta potentially containing helminth eggs, and the expansion of food supply chains and improved technologies for the production and inspection of food products. These control measures have resulted in a significant reduction in the distribution and transmission of helminth infections and have improved the overall living conditions and health outcomes of the Vietnamese citizens. However, the persistence of several helminth diseases continues in some endemic areas, especially where poverty is widespread and local traditions include the consumption of raw foods, especially fish and meats. This manuscript provides an overview of the helminth infection prevention and control programs conducted in Vietnam, their achieved results, learned lessons, and future works.
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Helmintiasis , Helmintos , Animales , Humanos , Vietnam/epidemiología , Suelo/parasitología , Escolaridad , Carne , Helmintiasis/tratamiento farmacológico , Helmintiasis/epidemiología , Helmintiasis/prevención & controlRESUMEN
Trending three-dimensional tissue engineering platforms developed via biofabrication and bioprinting of exocrine glands are on the rise due to a commitment to organogenesis principles. Nevertheless, a proper extracellular matrix (ECM) microarchitecture to harbor primary cells is yet to be established towards human salivary gland (SG) organogenesis. By using porcine submandibular gland (SMG) biopsies as a proof-of-concept to mimic the human SG, a new decellularized ECM bioassembly platform was developed herein with varying perfusions of sodium dodecyl sulfate (SDS) to limit denaturing events and ensure proper preservation of the native ECM biochemical niche. Porcine SMG biopsies were perfused with 0.01%, 0.1%, and 1% SDS and bio-assembled magnetically in porous polycarbonate track-etched (PCTE) membrane. Double-stranded DNA (dsDNA), cell removal efficiency, and ECM biochemical contents were analyzed. SDS at 0.1% and 1% efficiently removed dsDNA (< 50 ng/mg) and preserved key matrix components (sulfated glycosaminoglycans, collagens, elastin) and the microarchitecture of native SMG ECM. Bio-assembled SMG decellularized ECM (dECM) perfused with 0.1-1% SDS enhanced cell viability, proliferation, expansion confluency rates, and tethering of primary SMG cells during 7 culture days. Perfusion with 1% SDS promoted greater cell proliferation rates while 0.1% SDS supported higher acinar epithelial expression when compared to basement membrane extract and other substrates. Thus, this dECM magnetic bioassembly strategy was effective for decellularization while retaining the original ECM biochemical niche and promoting SMG cell proliferation, expansion, differentiation, and tethering. Altogether, these outcomes pave the way towards the recellularization of this novel SMG dECM in future in vitro and in vivo applications.
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Matriz Extracelular Descelularizada , Ingeniería de Tejidos , Porcinos , Humanos , Animales , Ingeniería de Tejidos/métodos , Matriz Extracelular/metabolismo , Glándulas Salivales , Fenómenos Magnéticos , Andamios del TejidoRESUMEN
Human umbilical cord lining epithelial cells [CLECs) are naïve in nature and can be ethically recovered from cords that are routinely discarded. The success of using oral mucosal epithelial cells for cornea defects hints at the feasibility of treating cutaneous wounds using non-native CLECs. Herein, we characterized CLECs using flow cytometry (FC) and skin organotypic cultures in direct comparison with skin keratinocytes (KCs). This was followed by wound healing study to compare the effects of CLEC application and the traditional use of human skin allografts (HSGs) in a porcine wound model. While CLECs were found to express all the epidermal cell markers probed, the major difference between CLECs and KCs lies in the level of expression (in FC analysis) as well as in the location of expression (of the epithelium in organotypic cultures) of some of the basal cell markers probed. On the pig wounds, CLEC application promoted accelerated healing with no adverse reaction compared to HSG use. Though CLECs, like HSGs, elicited high levels of local and systemic immune responses in the animals during the first week, these effects were tapered off more quickly in the CLEC-treated group. Overall, the in vivo porcine data point to the potential of CLECs as a non-native and safe source of cells to treat cutaneous wounds.
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Cordón Umbilical , Cicatrización de Heridas , Animales , Células Epiteliales/metabolismo , Humanos , Queratinocitos , Piel/metabolismo , PorcinosRESUMEN
BACKGROUND Since the initial COVID-19 cases in 2019, the pandemic has expanded globally. Clinical data showed that dexamethasone treatment at a dose of 6 mg daily for up to 10 days in hospitalized patients with COVID-19 who were receiving respiratory support decreased 28-day mortality in COVID-19 patients. Recent reports, on the other hand, have indicated that both steroid resistance and rebound events occur. We report a case of rebound inflammation after the termination of dexamethasone medication in a 38-year-old man with severe COVID-19 pneumonia, which improved after the reintroduction of dexamethasone. CASE REPORT A 38-year-old male patient with no past medical history of note presented with new onset of dyspnea. He was subsequently diagnosed with severe coronavirus disease 2019 (COVID-19). Initially, the patient was clinically improved following a 3-day course of 16 mg of dexamethasone daily. Shortly after discontinuing corticosteroids, the patient's clinical condition deteriorated, necessitating increased oxygen support. Following the reintroduction of corticosteroids, the patient gradually improved and responded favorably in terms of respiratory function, symptoms, and imaging, after which he was successfully discharged. CONCLUSIONS This case exemplifies the previously observed rebound effects of discontinuing dexamethasone medication in individuals with severe COVID-19 pneumonia. The timing and length of dexamethasone medication should be tailored to the individual patient. In addition, monitoring lung function should be part of the gradual withdrawal of dexamethasone to avoid rebound lung inflammation and the long-term effects of increasing lung fibrosis.
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Tratamiento Farmacológico de COVID-19 , Neumonía , Corticoesteroides , Adulto , Dexametasona/uso terapéutico , Humanos , Inflamación , Masculino , SARS-CoV-2RESUMEN
To determine the therapeutic efficacy of human umbilical cord lining mesenchymal stromal cells (CL-MSCs) (US Patent number 9,737,568) in lupus-prone MRL/lpr (Faslpr) mice and elucidate its working mechanisms. A total of 4 doses of (20-25) × 106 cells/kg of CL-MSCs was given to 16-week-old female Faslpr mice by intraperitoneal injection. Three subsequent doses were given on 17 weeks, 18 weeks, and 22 weeks, respectively. Six-week-old Faslpr mice were used as disease pre-onset controls. Mice were monitored for 10 weeks. Mouse kidney function was evaluated by examining complement component 3 (C3) deposition, urinary albumin-to-creatinine ratio (ACR), and lupus nephritis (LN) activity and chronicity. Working mechanisms were elucidated by flow cytometry, Luminex/ELISA (detection of anti-dsDNA and isotype antibodies), and RNA sequencing. CL-MSCs improved mice survival and kidney function by reducing LN activity and chronicity and lymphocyte infiltration over 10 weeks. CL-MSCs also reduced urinary ACR, renal complement C3 deposition, anti-dsDNA, and isotype antibodies that include IgA, IgG1, IgG2a, IgG2b, and IgM. Immune and cytokine profiling demonstrated that CL-MSCs dampened inflammation by suppressing splenic neutrophils and monocytes/macrophages, reducing plasma IL-6, IL-12, and CXCL1 and stabilizing plasma interferon-γ and TNF-α. RNA sequencing further showed that CL-MSCs mediated immunomodulation via concerted action of pro-proinflammatory cytokine-induced chemokines and production of nitric oxide in macrophages. CL-MSCs may provide a novel myeloid (neutrophils and monocytes/macrophages)-targeting therapy for SLE.
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Lupus Eritematoso Sistémico , Nefritis Lúpica , Células Madre Mesenquimatosas , Femenino , Humanos , Animales , Ratones , Ratones Endogámicos MRL lpr , Riñón/metabolismo , Citocinas/uso terapéutico , Inmunoglobulina G/uso terapéutico , Células Madre Mesenquimatosas/metabolismo , Cordón Umbilical/metabolismo , Lupus Eritematoso Sistémico/terapiaRESUMEN
In diabetes there is a long latency between the onset of hyperglycemia and the appearance of structural microangiopathy. Because Ly6Clow patrolling monocytes (PMo) behave as housekeepers of the vasculature, we tested whether PMo protect microvessels against diabetes. We found that in wild-type mice, diabetes reduced PMo in the general circulation but increased by fourfold the absolute number of PMo adherent to retinal vessels (leukostasis). Conversely, in diabetic NR4A1-/- mice, a model of absence of PMo, there was no increase in leukostasis, and at 6 months of diabetes, the number of retinal acellular capillaries almost doubled compared with diabetic wild-type mice. Circulating PMo showed gene expression changes indicative of enhanced migratory, vasculoprotective, and housekeeping activities, as well as profound suppression of genes related to inflammation and apoptosis. Promigratory CXCR4 was no longer upregulated at longer duration when retinal acellular capillaries begin to increase. Thus, after a short diabetes duration, PMo are the cells preferentially recruited to the retinal vessels and protect vessels from diabetic damage. These observations support the need for reinterpretation of the functional meaning of leukostasis in diabetes and document within the natural history of diabetic retinopathy processes of protection and repair that can provide novel paradigms for prevention.
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Diabetes Mellitus Experimental/complicaciones , Retinopatía Diabética/metabolismo , Monocitos/fisiología , Vasos Retinianos/patología , Animales , Regulación de la Expresión Génica/fisiología , Ratones , Ratones Noqueados , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismoRESUMEN
We investigated the safety of using umbilical cord-lining stem cells for liver regeneration and tested a novel method for stem cell delivery. Stem cells are known by their ability to repair damaged tissues and have the potential to be used as regenerative therapies. The umbilical cord's outer lining membrane is known to be a promising source of multipotent stem cells and can be cultivated in an epithelial cell growth medium to produce cell populations which possess the properties of both epithelial cells and embryonic stem cells-termed cord-lining epithelial cells (CLEC). Hepatocytes are epithelial cells of the liver and their proliferation upon injury is the main mechanism in restoring the liver. Earlier studies conducted showed CLEC can be differentiated into functioning hepatocyte-like cells (HLC) and can survive in immunologically competent specimens. In this study, we chose a porcine model to investigate CLEC as a treatment modality for liver failure. We selected 16 immune competent Yorkshire-Dutch Landrace pigs, with a mean weight of 40.5 kg, for this study. We performed a 50% hepatectomy to simulate the liver insufficient disease model. After the surgery, four pigs were transplanted with a saline scaffold while seven pigs were transplanted with a HLC scaffold. Five pigs died on the surgical table and were omitted from the study analysis. This study addressed the safety of transplanting human CLEC in a large animal model. The transplant interfaces were evaluated and no signs of cellular rejection were observed in both groups.
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Células Epiteliales/citología , Regeneración Hepática/fisiología , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre/citología , Animales , Células Cultivadas , Células Epiteliales/fisiología , Hepatocitos/citología , Hepatocitos/fisiología , Humanos , Células Madre/fisiología , PorcinosRESUMEN
Purpose: We investigated the use of human Cord Lining Mesenchymal Stem Cells (CL-MSCs) (US Patent number 9,737,568), in a rabbit hindlimb ischemia model, and evaluated their potential in stimulating neovascularization. Allogenic human CL- MSCs could potentially be used to treat patients with lower limb ischemia and non-healing wounds. Methods: Twenty rabbits were divided into two separate groups. We created a hindlimb ischemia model surgically. At 21 and 49 days post-operatively, animals in the treatment group were injected with CL-MSCs (500,000 cells per 0.2 ml on each site) at 10 different sites (Quadriceps- 4 sites, Hamstrings- 4 sites and Calf--2 sites) in the hindlimb muscles. The control group received only saline injection to the corresponding sites at the same time point as the treatment group. We then evaluated the effects of treatment on neovascularization by angiography, laser doppler perfusion imaging, as well as by histology. We evaluated the tissue samples for any signs of local immune reaction to the cell implantation. We also observed the rabbit clinically for any adverse effects after treatment. Results: We found a higher number of CD31 positive cells in the treatment group, with a greater number of capillaries found in the treated muscles. The Rectus Femoris demonstrated a median vessel count/muscle fiber of 0.121 for the treatment group, compared to 0.076 in the control group (median difference 0.04; 95% CI 0.001-0.11; p = 0.041). The Gastrocnemius demonstrated a median vessel count/muscle fiber of 0.175 for the treatment group, compared to 0.089 in the control group (median difference 0.087; 95% CI -0.006 to 0.234; p = 0.07). Blood perfusion quantification through Laser Doppler Perfusion Imaging (LDPI) also demonstrated a non-statistically significant increase in perfusion in favor of the treatment group. CL-MSCs demonstrated no toxicity associated morbidity and minimal local immune reaction to implantation. Conclusion: CL-MSCs have a positive effect on angiogenesis in a rabbit hindlimb ischemia model. This preliminary data is encouraging and paves the way for future large animal studies or for clinical trials.
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BACKGROUND: Persistent corneal epithelial defect (PED) is a consequence of many ocular surface disorders. Although many therapies have been suggested, the treatment of this disease have faced a lot of difficulties up to now. The transplatation of cultivated amniotic epithelial cells sheets is the new promised method for PED. Cord lining epithelial cells (CLECs) are epithelial cells of amniotic membrane of umbilical cord, so these cultivated cells sheet may be good for treating PED. AIM: To evaluate the efficacy of the transplantation of cultivated CLECs sheets in treatment of PED and analyze some influential factors of this therapy. METHODS: A prospective interventional case series with transplantation of tissue-cultured human CLECs in 37 PED eyes in Vietnam National Institute of Ophthalmology. RESULTS: Thirty four of 37 eyes were healed with the cells transplantation and 22 eyes of them healed within a week postoperatively. There were normal corneal scars and normal corneal epithelial cell (by impression cytology detection) on transplantation site in all 31 successful cases. The other successful eyes were done lamellar keratoplasty (respectively in 1 month, 3 months, 6 months and 27 months postoperatively) to investigate the histopathology of the CLECs transplant site. The histopathological images showed normal corneal scar and there was no appearance of CLECs in transplant site. CONCLUSION: tissue-cultured human CLECs transplantation is a quite safe and effective treatment for persistent corneal epithelial defect. The CLECs may help the epithelial healing at early stage but do not exist at transplant site for a long time.
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Iatrogenic adverse events in clinical trials of retroviral vector-mediated gene-corrected cells have prioritized the urgent need for more comprehensive and stringent assessment of potentially genotoxic off-target alterations and the biosafety of cells intended for therapeutic applications. Genome editing tools such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced palindromic repeats (CRISPR)-Cas9 nuclease systems are being investigated as safer and efficient alternatives for site-directed genome modification. Using site-specific integration into the AAVS1 locus of primary human cells as an example, we present an integrated approach to multimodal investigation of off-target alterations and an evaluation of potential genotoxicity induced by ZFN-mediated integration of a therapeutic transgene.
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Daño del ADN , Células Epiteliales/citología , Edición Génica , Ingeniería Genética/métodos , Transgenes , Cordón Umbilical/citología , Nucleasas con Dedos de Zinc/metabolismo , Células Cultivadas , Células Epiteliales/metabolismo , Vectores Genéticos , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Recombinación Genética , Transcriptoma , Cordón Umbilical/metabolismo , Nucleasas con Dedos de Zinc/genéticaRESUMEN
Costly coagulation factor VIII (FVIII) replacement therapy is a barrier to optimal clinical management of hemophilia A. Therapy using FVIII-secreting autologous primary cells is potentially efficacious and more affordable. Zinc finger nucleases (ZFN) mediate transgene integration into the AAVS1 locus but comprehensive evaluation of off-target genome effects is currently lacking. In light of serious adverse effects in clinical trials which employed genome-integrating viral vectors, this study evaluated potential genotoxicity of ZFN-mediated transgenesis using different techniques. We employed deep sequencing of predicted off-target sites, copy number analysis, whole-genome sequencing, and RNA-seq in primary human umbilical cord-lining epithelial cells (CLECs) with AAVS1 ZFN-mediated FVIII transgene integration. We combined molecular features to enhance the accuracy and activity of ZFN-mediated transgenesis. Our data showed a low frequency of ZFN-associated indels, no detectable off-target transgene integrations or chromosomal rearrangements. ZFN-modified CLECs had very few dysregulated transcripts and no evidence of activated oncogenic pathways. We also showed AAVS1 ZFN activity and durable FVIII transgene secretion in primary human dermal fibroblasts, bone marrow- and adipose tissue-derived stromal cells. Our study suggests that, with close attention to the molecular design of genome-modifying constructs, AAVS1 ZFN-mediated FVIII integration in several primary human cell types may be safe and efficacious.
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Endonucleasas/metabolismo , Factor VIII/genética , Estudio de Asociación del Genoma Completo , Mutagénesis Insercional , Dedos de Zinc , Sitios de Unión , Factor VIII/metabolismo , Expresión Génica , Marcación de Gen , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células K562 , Unión Proteica , TransgenesRESUMEN
PURPOSE: To determine the effectiveness of human umbilical cord-derived mucin-expressing cord lining epithelial cells (CLEC-muc) as feeder cells in a coculture system for the cultivation of human limbal stem cells. METHODS: Human CLEC-muc were cultured in PTTe-1 medium and treated with mitomycin C to arrest their growth to make the feeder layer. Single-cell suspension of limbal cells was prepared from corneal rim collected from the Singapore Eye Bank. Limbal cells were cultured in a coculture system with CLEC-muc as well as 3T3 cells as feeder layer. We compared the colony-forming efficiency and cell morphology of the limbal cells cultured in the two different feeder layers. We also compared the expression level of several putative limbal stem cell markers, such as HES1, ABCG2, ΔNP63, and BMI1, in the cultured limbal cells by immunostaining and quantitative (q)RT-PCR. Expression of cytokeratins CK14, CK15, CK19, CK3, and CK4 was further compared. RESULTS: Human limbal epithelial cells cultured in both types of feeder layers showed comparable cell morphology and colony-forming efficiency. These cells exhibited a similar expression pattern of HES1, ABCG2, ΔNP63, BMI1, CK14, CK15, CK19, and CK3 as detected by immunostaining and PCR. CONCLUSIONS: Human CLEC-muc may be a suitable alternative to conventional mouse 3T3 feeder cells, which may reduce the risk of zoonotic infection.
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Epitelio Corneal/citología , Limbo de la Córnea/citología , Células Madre/citología , Cordón Umbilical/citología , Biomarcadores/metabolismo , Recuento de Células , Línea Celular , Proliferación Celular , Técnicas de Cocultivo , ADN/genética , Epitelio Corneal/crecimiento & desarrollo , Epitelio Corneal/metabolismo , Células Nutrientes , Regulación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Humanos , Queratinas/biosíntesis , Queratinas/genética , Limbo de la Córnea/crecimiento & desarrollo , Limbo de la Córnea/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/metabolismoRESUMEN
Objective: Nanofibers for tissue scaffolding and wound dressings hold great potential in realizing enhanced healing of wounds in comparison with conventional counterparts. Previously, we demonstrated good fibroblast adherence and growth on a newly developed scaffold, Tegaderm™-Nanofiber (TG-NF), made from poly É-caprolactone (PCL)/gelatin nanofibers electrospun onto Tegaderm (TG). The purpose of this study is to evaluate the performance and safety of TG-NF dressings in partial-thickness wound in a pig healing model. Approach: To evaluate the rate of reepithelialization, control TG, human dermal fibroblast-seeded TG-NF(+) and -unseeded TG-NF(-) were randomly dressed onto 80 partial-thickness burns created on four female and four male pigs. Wound inspections and dressings were done after burns on day 7, 14, 21, and 28. On day 28, full-thickness biopsies were taken for histopathological evaluation by Masson-Trichrome staining for collagen and hematoxylin-eosin staining for cell counting. Results: No infection and severe inflammation were recorded. Wounds treated with TG-NF(+) reepithelialized significantly faster than TG-NF(-) and control. Wound site inflammatory responses to study groups were similar as total cell counts on granulation tissues show no significant differences. Most of the wounds completely reepithelialized by day 28, except for two wounds in control and TG-NF(-). A higher collagen coverage was also recorded in the granulation tissues treated with TG-NF(+). Innovation and Conclusion: With better reepithelialization achieved by TG-NF(+) and similar rates of wound closure by TG-NF(-) and control, and the absence of elevated inflammatory responses to TG-NF constructs, TG-NF constructs are safe and demonstrated good healing potentials that are comparable to Tegaderm.
RESUMEN
Intense scientific research over the past two decades has yielded much knowledge about embryonic stem cells, mesenchymal stem cells from bone marrow, as well as epithelial stem cells from the skin and cornea. However, the billions of dollars spent in this research have not overcome the fundamental difficulties intrinsic to these stem cell strains related to ethics (embryonic stem cells), as well as to technical issues such as accessibility, ease of cell selection and cultivation, and expansion/mass production, while maintaining consistency of cell stemness (all of the stem cell strains already mentioned). Overcoming these technical hurdles has made stem cell technology expensive and any potential translational products unaffordable for most patients. Commercialization efforts have been rendered unfeasible by this high cost. Advanced biomedical research is on the rise in Asia, and new innovations have started to overcome these challenges. The Nobel Prize-winning Japanese development of iPSCs has effectively introduced a possible replacement for embryonic stem cells. For non-embryonic stem cells, cord lining stem cells (CLSCs) have overcome the preexisting difficulties inherent to mesenchymal stem cells from the bone marrow as well as epithelial stem cells from the skin and cornea, offering a realistic, practical, and affordable alternative for tissue repair and regeneration. This novel CLSC technology was developed in Singapore in 2004 and has 22 international patents granted to date, including those from the US and UK. CLSCs are derived from the umbilical cord outer lining membrane (usually regarded as medical waste) and is therefore free from ethical dilemmas related to its collection. The large quantity of umbilical cord lining membrane that can be collected translates to billions of stem cells that can be grown in primary stem cell culture and therefore very rapid and inexpensive cell cultivation and expansion for clinical translational therapies. Both mesenchymal and epithelial stem cells can be isolated from the umbilical cord lining membrane, usefully regenerating not only mesenchymal tissue, such as bone, cartilage, and cardiac and striated muscle, but also epithelial tissue, such as skin, cornea, and liver. Both mesenchymal and epithelial CLSCs are immune privileged and resist rejection. Clinically, CLSCs have proved effective in the treatment of difficult-to-heal human wounds, such as diabetic ulcers, recalcitrant chronic wounds, and even persistent epithelial defects of the cornea. Heart and liver regeneration has been shown to be successful in animal studies and await human trials. CLSCs have also been shown to be an effective feeder layer for cord blood hematopoietic stem cells and, more recently, has been recognized as an abundant and high-quality source of cells for iPSC production. Banking of CLSCs by cord blood banks in both private and public settings is now available in many countries, so that individuals may have their personal stores of CLSCs for future translational applications for both themselves and their families. Cord lining stem cells are strongly positioned to be the future of cell therapy and regenerative medicine.
Asunto(s)
Células Epiteliales/citología , Células Madre Mesenquimatosas/citología , Cordón Umbilical/citología , Animales , Antígenos CD/metabolismo , Diferenciación Celular , Células Epiteliales/metabolismo , Cardiopatías/terapia , Humanos , Trasplante de Células Madre Mesenquimatosas , Medicina RegenerativaRESUMEN
Within the immunosuppressive ocular microenvironment, there are constitutively present the immunomodulating neuropeptides alpha-melanocyte stimulating hormone (α-MSH) and neuropeptide Y (NPY) that promote suppressor functionality in macrophages. In this study, we examined the possibility that α-MSH and NPY modulate phagocytic activity in macrophages. The macrophages treated with α-MSH and NPY were significantly suppressed in their capacity to phagocytize unopsonized Escherichia coli and Staphylococcus aureus bioparticles, but not antibody-opsonized bioparticles. The neuropeptides significantly suppressed phagolysosome activation, and the FcR-associated generation of reactive oxidative species as well. This suppression corresponds to neuropeptide modulation of macrophage functionality within the ocular microenvironment to suppress the activation of immunogenic inflammation.