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1.
Int J Mol Sci ; 20(15)2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31374809

RESUMO

Dry eye disease (DED) is a multifactorial syndrome that can be caused by alteration in the quality or quantity of the precorneal tear film. It is considered one of the most common ocular conditions leading patients to seek eye care. The current method for diagnostic evaluations and follow-up examinations of DED is a combination of clinical signs and symptoms determined by clinical tests and questionnaires, respectively. The application of powerful omics technologies has opened new avenues toward analysis of subjects in health and disease. Metabolomics is a new emerging and complementary research discipline to all modern omics in the comprehensive analysis of biological systems. The identification of distinct metabolites and integrated metabolic profiles in patients can potentially inform clinicians at an early stage or during monitoring of disease progression, enhancing diagnosis, prognosis, and the choice of therapy. In ophthalmology, metabolomics has gained considerable attention over the past decade but very limited such studies have been reported on DED. This paper aims to review the application of tear metabolomics in DED.


Assuntos
Síndromes do Olho Seco/metabolismo , Metaboloma , Metabolômica/métodos , Lágrimas/metabolismo , Animais , Síndromes do Olho Seco/diagnóstico , Humanos , Prognóstico
2.
Exp Eye Res ; 97(1): 1-9, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22342952

RESUMO

In patients with limbal stem cell deficiency (LSCD), transplantation of ex vivo expanded human limbal epithelial cells (HLECs) can restore the structural and functional integrity of the corneal surface. However, the protocol for cultivation and transplantation of HLECs differ significantly, and in most protocols growth additives such as cholera toxins, exogenous growth factors, hormones and fetal calf serum are used. In the present article, we compare for the first time human limbal epithelial cells (HLECs) cultivated on human amniotic membrane (HAM) in a complex medium (COM) including fetal bovine serum to a medium with human serum as single growth supplement (HSM), and report on our first examinations of HLECs expanded in autologous HSM and used for transplant procedures in patients with LSCD. Expanded HLECs were examined by genome-wide microarray, RT-PCR, Western blotting, and for cell viability, morphology, expression of immunohistochemical markers and colony forming efficiency. Cultivation of HLECs in HSM produced a multilayered epithelium where cells with markers associated with LESCs were detected in the basal layers. There were few transcriptional differences and comparable cell viability between cells cultivated in HSM and COM. The p63 gene associated with LESCs were expressed 3.5 fold more in HSM compared to COM, and Western blotting confirmed a stronger p63α band in HSM cultures. The cornea-specific keratin CK12 was equally found in both culture conditions, while there were significantly more CK3 positive cells in HSM. Cells in epithelial sheets on HAM remaining after transplant surgery of patients with LSCD expressed central epithelial characteristics, and dissociated cells cultured at low density on growth-arrested fibroblasts produced clones containing 21 ± 12% cells positive for p63α (n = 3). In conclusion, a culture medium without growth additives derived from animals or from animal cell cultures and with human serum as single growth supplement may serve as an equivalent replacement for the commonly used complex medium for ex vivo expansion of HLECs on HAM.


Assuntos
Âmnio , Células Epiteliais/citologia , Limbo da Córnea/citologia , Células-Tronco/citologia , Alicerces Teciduais , Animais , Biomarcadores/metabolismo , Sangue , Western Blotting , Bovinos , Técnicas de Cultura de Células , Sobrevivência Celular , Meios de Cultura , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Imuno-Histoquímica , Queratina-12/genética , Queratina-12/metabolismo , Limbo da Córnea/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Albumina Sérica , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
3.
J Cell Biochem ; 112(2): 684-93, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21268090

RESUMO

To obtain sufficient numbers of cells for tissue engineering applications, human bone marrow-derived mesenchymal stem cells (hBM-MSC) are commonly cultured as monolayers in incubators containing room air. In this study, we investigated whether three-dimensional (3D) culture conditions and incubator gas concentrations more similar to those observed in vivo impacted on cell expansion, differentiation capability, or phenotype of hBM-MSC. We found that 3D culture alone increased the expression of some molecules involved in osteogenic and adipogenic differentiation. In contrast, 3D culture did not induce chondrogenic differentiation, but enhanced the response to the chondrogenic differentiation medium. Changing the oxygen concentration to 6% and the carbon dioxide concentration to 7.5% did not impact on the results of any of our assays, showing that the hyperoxia of room air is not detrimental to hBM-MSC proliferation, differentiation, or phenotype.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Células-Tronco Mesenquimais/citologia , Diferenciação Celular/genética , Células Cultivadas , Citometria de Fluxo , Humanos , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
4.
Knee Surg Sports Traumatol Arthrosc ; 18(10): 1407-16, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20020100

RESUMO

Treatment of focal lesions of the articular cartilage of the knee using chondrocytes in a hyaluronic acid (HA) scaffold is already being investigated in clinical trials. An alternative may be to use mesenchymal stem cells (MSC). We have compared articular chondrocytes with MSC from human bone marrow (BM) and adipose tissue (AT), all cultured in HA scaffolds, for their ability to express genes and synthesize proteins associated with chondrogenesis. The cells were expanded in monolayer cultures. After seeding into the scaffold, the chondrocytes were maintained in medium, while the two MSC populations were given a chondrogenic differentiation medium. Chondrogenesis was assessed by real-time RT-PCR for chondrocyte-associated genes, by immunohistochemistry and by ELISA for collagens in the supernatant. Redifferentiation of the dedifferentiated chondrocytes in the HA scaffold was shown by a modest increase in type II collagen mRNA (COL2A1) and reduction in COL1A1. BM-MSC expressed 600-fold higher levels of COL2A1 than chondrocytes after 3 weeks in the scaffold. The levels of aggrecan (AGC1) and COL1A1 were similar for chondrocyte and BM-MSC scaffold cultures, while COL10A1 was higher in the BM-MSC. AT-MSC expressed levels of COL2A1 and COL1A1 similar to chondrocytes, but less AGC1 and COL10A1. Surprisingly, little collagen II protein was observed in the scaffold. Instead, collagen II was found in the culture medium. Chondrogenesis in HA scaffolds was more efficient using BM-MSC than AT-MSC or chondrocytes. Some of the secreted collagen II escaped entrapment in the extracellular space and was detected in the culture medium.


Assuntos
Células da Medula Óssea/metabolismo , Condrogênese/fisiologia , Células-Tronco Mesenquimais/metabolismo , Engenharia Tecidual/métodos , Alicerces Teciduais , Adulto , Análise de Variância , Células da Medula Óssea/citologia , Cartilagem Articular/cirurgia , Diferenciação Celular/fisiologia , Células Cultivadas , Estudos de Coortes , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Ácido Hialurônico/metabolismo , Imuno-Histoquímica , Masculino , Células-Tronco Mesenquimais/citologia , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto Jovem
5.
Head Neck ; 42(8): 2088-2098, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32202693

RESUMO

BACKGROUND: We previously showed a tumor-suppressive function of S100A14 in oral squamous cell carcinoma (OSCC). This study aimed to examine the prognostic significance and differentiation-related function of S100A14 in OSCC. METHODS: S100A14 expression was examined in 170 OSCCs from Norwegian and Nepalese populations using immunohistochemistry. Pro-differentiation function was investigated by overexpressing and silencing S100A14 expression in OSCC-derived cells. External transcriptomic datasets were used to validate association between S100A14 and differentiation markers in OSCC. RESULT: Loss of S100A14 expression at the invading tumor fronts significantly correlated with poor differentiation and reduced 10-years survival of OSCC-patients. Multivariate Cox analysis identified S100A14 to be an independent prognostic factor. Modulation of S100A14 expression in OSCC-derived cells positively correlated with the expression of differentiation markers. Analysis of external datasets supported the pro-differentiation function of S100A14. CONCLUSION: These results indicate that S100A14 is a pro-differentiation protein and its expression might be useful as a prognostic marker in OSCC.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Diferenciação Celular , Linhagem Celular Tumoral , Humanos , Neoplasias Bucais/genética , Prognóstico , Carcinoma de Células Escamosas de Cabeça e Pescoço
6.
PLoS One ; 15(12): e0243914, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33326470

RESUMO

PURPOSE: Seeking to improve the access to regenerative medicine, this study investigated the structural and transcriptional effects of storage temperature on human oral mucosal epithelial cells (OMECs). METHODS: Cells were stored at four different temperatures (4°C, 12°C, 24°C and 37°C) for two weeks. Then, the morphology, cell viability and differential gene expression were examined using light and scanning electron microscopy, trypan blue exclusion test and TaqMan gene expression array cards, respectively. RESULTS: Cells stored at 4°C had the most similar morphology to non-stored controls with the highest viability rate (58%), whereas the 37°C group was most dissimilar with no living cells. The genes involved in stress-induced growth arrest (GADD45B) and cell proliferation inhibition (TGFB2) were upregulated at 12°C and 24°C. Upregulation was also observed in multifunctional genes responsible for morphology, growth, adhesion and motility such as EFEMP1 (12°C) and EPHA4 (4°C-24°C). Among genes used as differentiation markers, PPARA and TP53 (along with its associated gene CDKN1A) were downregulated in all temperature conditions, whereas KRT1 and KRT10 were either unchanged (4°C) or downregulated (24°C and 12°C; and 24°C, respectively), except for upregulation at 12°C for KRT1. CONCLUSIONS: Cells stored at 12°C and 24°C were stressed, although the expression levels of some adhesion-, growth- and apoptosis-related genes were favourable. Collectively, this study suggests that 4°C is the optimal storage temperature for maintenance of structure, viability and function of OMECs after two weeks.


Assuntos
Proliferação de Células/fisiologia , Células Epiteliais/fisiologia , Mucosa Bucal/fisiologia , Manejo de Espécimes , Apoptose/fisiologia , Adesão Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Criopreservação , Humanos , Temperatura
7.
Int J Dev Biol ; 52(8): 1033-42, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18956336

RESUMO

Culture of mesenchymal stem cells (MSCs) under conditions promoting proliferation and differentiation, while supporting genomic and epigenetic stability, is essential for therapeutic use. We report here the extent of genome-wide DNA gains and losses and of DNA methylation instability on 170 cancer-related promoters in bone marrow (BM) MSCs during culture to late passage in medium containing fetal bovine serum (FBS) or autologous serum (AS). Comparative genomic hybridization indicates that expansion of BMMSCs elicits primarily telomeric deletions in a subpopulation of cells, the extent of which varies between donors. However, late passage cultures in AS consistently display normal DNA copy numbers. Combined bisulfite restriction analysis and bisulfite sequencing show that although DNA methylation states are overall stable in culture, AS exhibits stronger propensity than FBS to maintain unmethylated states. Comparison of DNA methylation in BMMSCs with freshly isolated and cultured adipose stem cells (ASCs) also reveals that most genes unmethylated in both BMMSCs and ASCs in early passage are also unmethylated in uncultured ASCs. We conclude that (i) BMMSCs expanded in AS or FBS may display localized genetic alterations, (ii) AS tends to generate more consistent genomic backgrounds and DNA methylation patterns, and (iii) the unmethylated state of uncultured MSCs is more likely to be maintained in culture than the methylated state.


Assuntos
Meios de Cultura , Epigênese Genética , Instabilidade Genômica , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Bovinos , Técnicas de Cultura de Células , Hibridização Genômica Comparativa , Metilação de DNA , Dosagem de Genes , Humanos , Especificidade da Espécie
8.
Cells ; 8(3)2019 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-30875861

RESUMO

Hyaluronan (HA), also termed hyaluronic acid or hyaluronate, is a major component of the extracellular matrix. This non-sulfated glycosaminoglycan plays a key role in cell proliferation, growth, survival, polarization, and differentiation. The diverse biological roles of HA are linked to the combination of HA's physicochemical properties and HA-binding proteins. These unique characteristics have encouraged the application of HA-based hydrogel scaffolds for stem cell-based therapy, a successful method in the treatment of limbal stem cell deficiency (LSCD). This condition occurs following direct damage to limbal stem cells and/or changes in the limbal stem cell niche microenvironment due to intrinsic and extrinsic insults. This paper reviews the physical properties, synthesis, and degradation of HA. In addition, the interaction of HA with other extracellular matrix (ECM) components and receptor proteins are discussed. Finally, studies employing HA-based hydrogel scaffolds in the treatment of LSCD are reviewed.


Assuntos
Ácido Hialurônico/farmacologia , Hidrogéis/farmacologia , Limbo da Córnea/citologia , Transplante de Células-Tronco , Alicerces Teciduais/química , Matriz Extracelular/metabolismo , Humanos , Ácido Hialurônico/química
9.
Bioengineering (Basel) ; 6(4)2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31652804

RESUMO

Hyaluronan (HA), a major component of the extracellular matrix, plays a key role in cell proliferation, growth, survival, polarization and differentiation. We investigated the optimization of a HA hydrogel scaffold for culture of human oral mucosal epithelial cells (OMECs) for potential application in limbal stem cell therapy. The effect of the optimized scaffold on OMEC cell sheet morphology, cell metabolic activity and expression of genes associated with stemness, adherence and cell damage was studied. The results indicate that HA hydrogels crosslinked with polyethylene glycol diacrylate (PEGDA) failed to support OMEC attachment and growth. However, HA hydrogel scaffolds dried for three days and coated with 1 mg/mL collagen IV produced a full OMEC sheet. Cell morphology was comparable to control after three weeks culture, maintaining 76% metabolic activity. Of apoptosis-related genes, the pro-apoptotic markers CASP3 and BAX2 were upregulated and downregulated, respectively, compared to control whereas the anti-apoptotic marker BCL2 was downregulated. The expression level of stemness genes ΔNp63α and ABCG2 was significantly higher than control. Genes associated with improved scar-less wound healing (integrin-V) and protection of the ocular surface (cadherin-1) had ~3-fold increased expression. These data suggest that our optimized HA-hydrogel scaffold could enhance culture of OMEC cell sheets for use in ocular reconstruction.

10.
Bone ; 43(4): 700-7, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18620088

RESUMO

The Ca(2+)/Calmodulin-dependent protein kinase (CaMK) family is activated in response to elevation of intracellular Ca(2+), and includes CaMK1 (as well as CaMK2 and CaMK4), which exists as different isoforms (alpha, beta, gamma and delta). CaMK1 is present in several cell types and may be involved in various cellular processes, but its role in bone is unknown. In situ hybridization was used to determine the spatial and temporal expression of CaMK1beta during endochondral bone development in mouse embryos and newborn pups. The cellular and subcellular distribution of CaMK1 was assessed by quantitative immunogold electron microscopy (EM). The role of CaMK1beta in mouse calvarial osteoblasts was investigated by using small interfering RNA (siRNA) to silence its expression, while in parallel monitoring cell proliferation and levels of skeletogenic transcripts. cRNA in situ hybridization and EM studies show that CaMK1beta is mainly located in developing long bones and vertebrae (from ED14.5 until day 10 after birth), with highest expression in epiphyseal growth plate hypertrophic chondrocytes. By RT-PCR, we show that CaMK1beta2 (but not beta1) is expressed in mouse hind limbs (in vivo) and mouse calvarial osteoblasts (in vitro), and also in primary human articular chondrocyte cultures. Silencing of CaMK1beta in mouse calvarial osteoblasts by siRNA significantly decreases osteoblast proliferation and c-Fos gene expression (approx. 50%), without affecting skeletogenic markers for more differentiated osteoblasts (i.e. Cbfa1/Runx2, Osterix (Osx), Osteocalcin (Oc), Alkaline phosphatase (Alp) and Osteopontin (Opn)). These results identify CaMK1beta as a novel regulator of osteoblast proliferation, via mechanisms that may at least in part involve c-Fos, thus implicating CaMK1beta in the regulation of bone and cartilage development.


Assuntos
Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/metabolismo , Proliferação de Células , Lâmina de Crescimento/metabolismo , Osteoblastos/metabolismo , Animais , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Osso e Ossos/ultraestrutura , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/genética , Células Cultivadas , Condrócitos/citologia , Condrócitos/enzimologia , Condrócitos/metabolismo , Epífises/embriologia , Epífises/enzimologia , Epífises/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Lâmina de Crescimento/embriologia , Lâmina de Crescimento/enzimologia , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Osteoblastos/citologia , Osteoblastos/enzimologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Crânio/citologia
11.
Mol Biol Cell ; 16(3): 1131-41, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15635089

RESUMO

Stromal stem cells proliferate in vitro and may be differentiated along several lineages. Freshly isolated, these cells have been too few or insufficiently pure to be thoroughly characterized. Here, we have isolated two populations of CD45-CD34+CD105+ cells from human adipose tissue which could be separated based on expression of CD31. Compared with CD31+ cells, CD31- cells overexpressed transcripts associated with cell cycle quiescence and stemness, and transcripts involved in the biology of cartilage, bone, fat, muscle, and neural tissues. In contrast, CD31+ cells overexpressed transcripts associated with endothelium and the major histocompatibility complex class II complex. Clones of CD31- cells could be expanded in vitro and differentiated into cells with characteristics of bone, fat, and neural-like tissue. On culture, transcripts associated with cell cycle quiescence, stemness, certain cytokines and organ specific genes were down-regulated, whereas transcripts associated with signal transduction, cell adhesion, and cytoskeletal +CD105+CD31- cells from human adipose tissue have stromal stem cell properties which may make them useful for tissue engineering.


Assuntos
Técnicas de Cultura de Células/métodos , Regulação da Expressão Gênica , Células-Tronco/citologia , Células Estromais/citologia , Transcrição Gênica , Tecido Adiposo/metabolismo , Antígenos CD , Antígenos CD34/biossíntese , Adesão Celular , Diferenciação Celular , Linhagem da Célula , Membrana Celular/metabolismo , Proliferação de Células , Células Cultivadas/metabolismo , Citoesqueleto/metabolismo , Regulação para Baixo , Endoglina , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Antígenos Comuns de Leucócito/biossíntese , Mesoderma/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , Receptores de Superfície Celular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Distribuição Tecidual , Engenharia Tecidual , Regulação para Cima , Molécula 1 de Adesão de Célula Vascular/biossíntese
12.
Methods Mol Biol ; 325: 35-46, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16761717

RESUMO

The stromal compartment of mesenchymal tissues is thought to harbor stem cells that display extensive proliferative capacity and multilineage potential. Stromal stem cells offer a potentially large therapeutic potential in the field of regenerative medicine. Adipose tissue contains a large number of stromal stem cells, is relatively easy to obtain in large quantities, and thus constitutes a very convenient source of stromal stem cells. Importantly, the number of stem cells obtained is compatible with extensive analyses of the cells in an uncultured, freshly isolated, form. This chapter describes procedures for isolating millions of highly purified stromal stem cells from human adipose tissue and methods of establishing polyclonal and monoclonal cultures of adipose tissue-derived stem cells.


Assuntos
Tecido Adiposo/citologia , Técnicas de Cultura de Células/métodos , Células-Tronco/citologia , Células Estromais/citologia , Diferenciação Celular , Separação Celular , Células Cultivadas , Colagenases/metabolismo , Eritrócitos/citologia , Humanos , Antígenos Comuns de Leucócito/biossíntese , Leucócitos/citologia , Mesoderma/patologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese
13.
J Funct Biomater ; 6(3): 917-45, 2015 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-26378588

RESUMO

Corneal endothelium is a single layer of specialized cells that lines the posterior surface of cornea and maintains corneal hydration and corneal transparency essential for vision. Currently, transplantation is the only therapeutic option for diseases affecting the corneal endothelium. Transplantation of corneal endothelium, called endothelial keratoplasty, is widely used for corneal endothelial diseases. However, corneal transplantation is limited by global donor shortage. Therefore, there is a need to overcome the deficiency of sufficient donor corneal tissue. New approaches are being explored to engineer corneal tissues such that sufficient amount of corneal endothelium becomes available to offset the present shortage of functional cornea. Although human corneal endothelial cells have limited proliferative capacity in vivo, several laboratories have been successful in in vitro expansion of human corneal endothelial cells. Here we provide a comprehensive analysis of different substrates employed for in vitro cultivation of human corneal endothelial cells. Advances and emerging challenges with ex vivo cultured corneal endothelial layer for the ultimate goal of therapeutic replacement of dysfunctional corneal endothelium in humans with functional corneal endothelium are also presented.

14.
Acta Ophthalmol ; 91(8): 769-75, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22937779

RESUMO

PURPOSE: Presently, our clinic is the only centre in Scandinavia that offers patients with corneal surface pathology including limbal stem cell deficiency (LSCD) transplantation of ex vivo expanded limbal epithelial cells (LECs). We here present clinical data of the first nine patients with LSCD who were transplanted with autologous LECs expanded in medium completely free of any animal-derived products and non-human/recombinant growth factors (including Cholera Toxin), and with autologous human serum as the only growth supplement. METHODS: We conducted a noncomparative retrospective study of patients with LSCD at our centre between 2009 and 2011. The diagnosis was based on history and clinical signs. A biopsy was taken from healthy limbus, and the epithelium was expanded on amniotic membrane (AM) in medium containing autologous serum and subsequently transplanted to the affected eye. RESULTS: Successful outcome was defined as relief of pain and photophobia and/or improved best corrected visual acuity (BCVA) and/or reestablishment of a stable corneal epithelium and regression of corneal vascularization. Five of the nine transplanted patients (55.6%) had an improvement in either subjective symptoms or objective findings (11- to 28-month follow-up). CONCLUSIONS: Our clinical study shows that patients with LSCD can be treated successfully with transplantation of LECs expanded ex vivo in a medium with autologous serum as the only growth supplement. The use of this novel culture system, which is devoid of animal-derived products and non-human/recombinant growth factors (including Cholera Toxin), reduces the risks of inter-species disease transmission and host immune responses to xenogenic proteins, both obvious advantages for the patient.


Assuntos
Técnicas de Cultura de Células/métodos , Doenças da Córnea/cirurgia , Epitélio Corneano/citologia , Limbo da Córnea/citologia , Soro/fisiologia , Transplante de Células-Tronco , Adulto , Idoso , Transplante de Células/métodos , Criança , Doenças da Córnea/fisiopatologia , Meios de Cultura , Células Epiteliais/citologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Transplante Autólogo , Resultado do Tratamento , Acuidade Visual/fisiologia , Adulto Jovem
15.
Acta Ophthalmol ; 91(3): 219-25, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-22429721

RESUMO

PURPOSE: Storage time for donor corneas in Optisol GS is limited compared to Eye Bank Organ Culture (EBOC). We here examine the epithelium on donor corneoscleral rims after primary storage in Optisol GS and subsequent incubation in EBOC. METHODS: Morphology was monitored by light and electron microscopy, expression of phenotypic and genotypic markers by immunohistochemistry and RT-PCR and changes in oxidative lipid and DNA damage by ELISA and COMET assay. RESULTS: A prominent loss of cells was observed after storage in Optisol GS. After maintenance in EBOC, spreading apical cells were Occludin(+) , while the staining for E-cadherin and Connexin-43 was less intense. There were an upregulation of Occludin and a downregulation of E-cadherin and Connexin-43. Eye Bank Organ Culture was associated with an ongoing proliferative activity and a downregulation of putative progenitor/stem cell marker ABCG2 and p63. Staining for 8-OHdG and Caspase-3 did not increase, while levels of malondialdehyde and number of DNA strand breaks and oxidized bases increased. CONCLUSIONS: This dual procedure should be pursued as an option to increase the storage time and the pool of available donor corneas. The observed downregulation of markers associated with stemness during EBOC is relevant considering the potential use of donor epithelium in the treatment of ocular surface disorders.


Assuntos
Sulfatos de Condroitina/uso terapêutico , Córnea , Criopreservação/métodos , Meios de Cultura Livres de Soro , Dextranos/uso terapêutico , Bancos de Olhos/métodos , Gentamicinas/uso terapêutico , Soluções para Preservação de Órgãos/uso terapêutico , Preservação de Órgãos/métodos , Biomarcadores/metabolismo , Proliferação de Células , Ensaio Cometa , Misturas Complexas/uso terapêutico , Dano ao DNA , Ensaio de Imunoadsorção Enzimática , Epitélio Corneano/metabolismo , Epitélio Corneano/ultraestrutura , Feminino , Humanos , Marcação In Situ das Extremidades Cortadas , Peroxidação de Lipídeos , Peróxidos Lipídicos/genética , Peróxidos Lipídicos/metabolismo , Masculino , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos , Estresse Oxidativo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Doadores de Tecidos
16.
Cell Transplant ; 21(8): 1697-709, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22410280

RESUMO

Cell therapy is a promising treatment modality to improve heart function in acute myocardial infarction. However, the mechanisms of action and the most suitable cell type have not been finally determined. We performed a study to compare the effects of mesenchymal stem cells (MSCs) harvested from different tissues on LV function and explore their effects on tissue structure by morphometry and histological staining for species and lineage relationship. MSCs from skeletal muscle (SM-MSCs) and adipose tissue (ADSCs) were injected in the myocardium of nude rats 1 week after myocardial infarction. After 4 weeks of observation, LVEF was significantly improved in the SM-MSCs group (39.1%) and in the ADSC group (39.6%), compared to the placebo group (31.0%, p < 0.001 for difference in change between groups). Infarct size was smaller after cell therapy (16.3% for SM-MSCs, 15.8% for ADSCs vs. 26.0% for placebo, p < 0.001), and the amount of highly vascularized granulation tissue in the border zone was significantly increased in both groups receiving MSCs (18.3% for SM-MSCs, 22.6% for ADSCs vs. 13.1% for placebo, p = 0.001). By in situ hybridization, moderate engraftment of transplanted cells was found, but no transdifferentiation to cardiomyocytes, endothelial cells, or smooth muscle cells was observed. We conclude that MSC injections lead to improved LVEF after AMI in rats predominantly by reduction of infarct size. After 4 weeks, we observed modulation of scar formation with significant increase in granulation tissue. Transdifferentiation of MSCs to cardiomyocytes or vascular cells did not contribute significantly in this process. MSCs from skeletal muscle and adipose tissue had similar effects.


Assuntos
Cicatriz/patologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Infarto do Miocárdio/terapia , Função Ventricular Esquerda/fisiologia , Doença Aguda , Tecido Adiposo/citologia , Animais , Transdiferenciação Celular , Ecocardiografia , Proteínas da Matriz Extracelular/metabolismo , Humanos , Músculo Esquelético/citologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , PPAR gama/metabolismo , Proteoglicanas/metabolismo , Ratos , Ratos Nus
17.
Tissue Eng Part C Methods ; 17(2): 219-27, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20799885

RESUMO

In this study we have isolated human primary uncultured articular chondrocytes. When these cells are allowed to proliferate within their own extracellular matrix (ECM), they begin to produce hyaline ECM molecules similar to embryological chondroblasts. These cells are called chondroblast-like cells. Upon continued culture these cells spread onto the plastic surface and dedifferentiate. We have characterized these three stages of chondral cells by gene expression and expression of microRNAs (miRNAs) and proteins. Gene expression was quantified by real-time reverse transcriptase (RT) polymerase chain reaction, miRNA expression by miRNA arrays, and protein synthesis by extra- and intracellular flow cytometry. Many of the genes, miRNAs, and proteins were differentially expressed in the different stages of chondral cells. In the context of cellular therapy, expression of some genes is a cause for concern. The best source of cells for treatment of lesions of hyaline cartilage has not yet been identified. Adult chondroblast-like cells may be strong candidates. Profound understanding of how expression of genes and synthesis of proteins are regulated in these cells, for instance, by miRNAs, may reveal new strategies for improving their synthesis of hyaline ECM. This insight is important to be able to use these cells in the clinic.


Assuntos
Cartilagem Articular/citologia , Desdiferenciação Celular , Condrócitos/citologia , Condrócitos/metabolismo , Regulação da Expressão Gênica , MicroRNAs/genética , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Proteínas da Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Humanos , MicroRNAs/metabolismo , Fenótipo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
Tissue Eng Part A ; 15(7): 1763-73, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19115828

RESUMO

Human mesenchymal stem cells (MSC) are popular candidates for tissue engineering. MSC are defined by their properties in two-dimensional (2D) culture systems. Cells in 2D are known to differ from their in vivo counterparts in cell shape, proliferation, and gene expression. Little is so far known about the phenotype and gene expression of cells in three-dimensional (3D) culture systems. To begin to unravel the impact of 3D versus 2D culture conditions on MSC, we have established MSC from adipose tissue and bone marrow in 3D cultures in alginate beads covalently modified with the tripeptide arginine-glycine-aspartic acid (RGD), the integrin-binding motif found in several molecules within the extracellular matrix. The MSC changed from their fibroblastoid shape (2D) to a small, compact shape when embedded in RGD alginate (3D). High viability was maintained throughout the experiment. The MSC retained expression of integrins known to bind RGD, and practically ceased to proliferate. Microarray analysis revealed that the gene expression in cells in RGD alginate was different both from the cells cultured in 2D and from prospectively isolated, uncultured MSC, but more similar to 2D cells. As alginate may be entirely dissolved, leaving the cells as single cell suspensions for various analyses, this represents a useful model for the study of cells in 3D cultures.


Assuntos
Alginatos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Alicerces Teciduais , Adolescente , Adulto , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Forma Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Imobilizadas/citologia , Células Imobilizadas/efeitos dos fármacos , Células Imobilizadas/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Ácido Glucurônico/farmacologia , Ácidos Hexurônicos/farmacologia , Humanos , Integrinas/metabolismo , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Oligopeptídeos/farmacologia , Fenótipo , Plásticos , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
19.
Tissue Eng Part A ; 14(12): 1999-2007, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18636947

RESUMO

Articular chondrocytes (AC) expanded in vitro for tissue engineering rapidly turn off collagen type II (COL2) synthesis. We wanted to inhibit this process sufficiently to obtain therapeutically useful numbers of AC without losing COL2 synthesis. To this end, AC were expanded on their own extracellular matrix (ECM) in structures designated chondrocytes in autologous ECM (CA-ECM). Here, AC maintained a rounded shape and proliferated rapidly. After 13-15 days in culture, 40 x 10(6) cells (median) could be obtained from a cartilage biopsy. Real-time RT-PCR showed a reduced, but persistent, production of COL2A1 mRNA at this time. Flow cytometry showed high levels of intracellular COL2, and immunogold electron microscopy showed high density of well-organized COL2 fibrils in newly synthesized ECM. Interestingly, high levels of COL1A1 mRNA and intracellular protein were detected, but no COL1 was found in the ECM. The slow loss of COL2A1 mRNA was paralleled by a loss of the COL2 regulating transcription factor SOX9 mRNA. Chromatin immunoprecipitation assays could not identify epigenetic histone modifications that would explain the observed changes in COL2 synthesis. Thus, the CA-ECM strategy allows AC to proliferate to clinically useful numbers while maintaining COL2 synthesis and secretion. This strategy may improve tissue engineering of joint surfaces.


Assuntos
Cartilagem Articular/citologia , Condrócitos/citologia , Colágeno Tipo II/biossíntese , Colágeno Tipo II/metabolismo , Adulto , Cartilagem Articular/metabolismo , Proliferação de Células , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/ultraestrutura , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Colágeno Tipo II/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Proteínas da Matriz Extracelular/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica , Histonas/metabolismo , Humanos , Espaço Intracelular/metabolismo , Pessoa de Meia-Idade , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética
20.
Stem Cells ; 23(9): 1357-66, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16081661

RESUMO

Human bone marrow mesenchymal stem cells (hMSCs) represent an appealing source of adult stem cells for cell therapy and tissue engineering, as they are easily obtained and expanded while maintaining their multilineage differentiation potential. All current protocols for in vitro culture of hMSCs include fetal bovine serum (FBS) as nutritional supplement. FBS is an undesirable additive to cells that are expanded for therapeutic purposes in humans because the use of FBS carries the risk of transmitting viral and prion diseases and proteins that may initiate xenogeneic immune responses. In the present study, we have therefore investigated if autologous serum (AS) or allogeneic human serum (alloHS) could replace FBS for the expansion of hMSCs in vitro. We discovered that the choice of serum affected hMSCs at several different levels. First, hMSCs in AS proliferated markedly faster than hMSCs in FBS, whereas use of alloHS resulted in hMSC growth arrest and death. Second, hMSCs in FBS differentiated more rapidly toward mesenchymal lineages compared with hMSCs in AS. Interestingly, genome-wide microarray analysis identified several transcripts involved in cell cycle and differentiation that were differentially regulated between hMSCs in FBS and AS. Finally, several transcripts, including some involved in cell cycle inhibition, were upregulated in hMSCs in FBS at a late passage, whereas the hMSC transcriptome in AS was remarkably stable. Thus, hMSCs may be expanded rapidly and with stable gene expression in AS in the absence of growth factors, whereas FBS induces a more differentiated and less stable transcriptional profile.


Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Mesenquimais/citologia , Animais , Células da Medula Óssea/citologia , Bovinos , Diferenciação Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Meios de Cultura , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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