Protocol to cryopreserve and isolate nuclei from adipose tissue without dimethyl sulfoxide.

Cryopreservation injuries involve nuclear DNA damage. A protocol for cryopreserving and isolating adipocyte nuclei is proposed. Adipose tissue samples were directly analyzed (NoCRYO-0h), or stored at -196°C for 7 days without 10% dimethyl sulfoxide (DMSO) (CRYO-WO-DMSO) or with DMSO (CRYO-W-DMSO). To determine the effect of DMSO on cryopreservation treatment, adipose tissue samples were stored at 4°C for 24 h with 10% DMSO (NoCRYO-W-DMSO-24h) and without (NoCRYO-WO-DMSO-24h). Samples were processed in isolation buffer, and nuclear integrity was measured by flow cytometry. The coefficient of variation, forward scatter, side scatter, and number of nuclei analyzed were evaluated. Pea (Pisum sativum) was used to measure the amount of DNA. All groups contained similar amounts of DNA to previously reported values and a satisfactory number of nuclei were analyzed. CRYO-W-DMSO presented a higher coefficient of variation (3.19 ± 0.09) compared to NoCRYO-0h (1.85 ± 0.09) and CRYO-WO-DMSO (2.02 ± 0.02). The coefficient of variation was increased in NoCRYO-W-DMSO-24h (3.80 ± 0.01) compared to NoCRYO-WO-DMSO-24h (2.46 ± 0.03). These results relate DMSO presence to DNA damage independently of the cryopreservation process. CRYO-W-DMSO showed increased side scatter (93.46 ± 5.03) compared to NoCRYO-0h (41.13 ± 3.19) and CRYO-WO-DMSO (48.01 ± 2.28), indicating that cryopreservation with DMSO caused chromatin condensation and/or nuclear fragmentation. CRYO-W-DMSO and CRYO-WO-DMSO presented lower forward scatter (186.33 ± 9.33 and 196.89 ± 26.86, respectively) compared to NoCRYO-0h (322.80 ± 3.36), indicating that cryopreservation reduced nuclei size. Thus, a simple method for cryopreservation and isolation of adipocyte nuclei causing less damage to DNA integrity was proposed.

Human immature dental pulp stem cells share key characteristic features with limbal stem cells.

OBJECTIVES: Limbal stem cells (LSC) are self-renewing, highly proliferative cells in vitro, which express a set of specific markers and in vivo have the capacity to reconstruct the entire corneal epithelium in cases of ocular surface injury. Currently, LSC transplantation is a commonly used procedure in patients with either uni- or bilateral total limbal stem cells deficiency (TLSCD). Although LSC transplantation holds great promise for patients, several problems need to be overcome. In order to find an alternative source of cells that can partially substitute LSC in cornea epithelium reconstruction, we aimed at investigating whether human immature dental pulp stem cells (hIDPSC) would present similar key characteristics as LSC and whether they could be used for corneal surface reconstruction in a rabbit TLSCD model. MATERIALS: We used hIDPSC, which co-express mesenchymal and embryonic stem cell markers and present the capacity to differentiate into derivative cells of the three germinal layers. TLSCD was induced by chemical burn in one eye of rabbits. After 30 days, the opaque tissue formed was removed by superficial keratectomy. Experimental group received undifferentiated hIDPSC, while control group only received amniotic membrane (AM). Both groups were sacrificed after 3 months. RESULTS AND CONCLUSIONS: We have demonstrated, using immunohistochemistry and reverse transcription-polymerase chain reaction, that hIDPSCs express markers in common with LSC, such as ABCG2, integrin beta1, vimentin, p63, connexin 43 and cytokeratins 3/12. They were also capable of reconstructing the eye surface after induction of unilateral TLSCD in rabbits, as shown by morphological and immunohistochemical analysis using human-specific antibodies against limbal and corneal epithelium. Our data suggest that hIDPSCs share similar characteristics with LSC and might be used as a potential alternative source of cells for corneal reconstruction.

Effect of polymorphisms of the MTHFR and APOE genes on susceptibility to diabetes and severity of diabetic retinopathy in Brazilian patients.

Diabetes mellitus (DM) is a highly prevalent complex genetic disorder. There has been a worldwide effort in the identification of susceptibility genes for DM and its complications, and the 5-10-methylenetetrahydrofolate reductase (MTHFR) and apolipoprotein-E (APOE) genes have been considered good candidate susceptibility genes to this condition. The objectives of the present study were to determine if the 677T MTHFR and epsilon2/epsilon3/epsilon4 APOE alleles are risk factors for DM and for severity of diabetic retinopathy (DR). A total of 248 individuals were studied: 107 healthy individuals and 141 diabetic patients (46 with type 1 diabetes and 95 with type 2 diabetes), who also had DR (81 with non-proliferative DR and 60 with proliferative DR). The polymorphisms were analyzed by PCR followed by digestion with restriction enzyme or the single-nucleotide primer extension method. No evidence of association between the 677TT genotype of MTHFR gene and DM [cases: TT = 10/95 (10.6%); controls: TT = 14/107 (13%)] or with severity of DR was observed [cases: TT = 5/60 (8.5%); controls: TT = 9/81 (11.1%); P > 0.05]. We also did not find evidence of an association between APOE alleles and proliferative DR (epsilon2, epsilon3 and epsilon4 in cases: 9, 76, and 15%, and in controls: 5, 88, and 12%, respectively) but the carriers of epsilon2 allele were more frequent among patients with type 2 DM and DR than in controls [cases: 15/95 (15.8%); controls: 7/107 (6.5%); P < 0.05]. Therefore, our results suggest that the epsilon2 allele/APOE might be a risk factor for diabetes in the Brazilian population.

Effect of polymorphisms of the MTHFR and APOE genes on susceptibility to diabetes and severity of diabetic retinopathy in Brazilian patients

Diabetes mellitus (DM) is a highly prevalent complex genetic disorder. There has been a worldwide effort in the identification of susceptibility genes for DM and its complications, and the 5-10-methylenetetrahydrofolate reductase (MTHFR) and apolipoprotein-E (APOE) genes have been considered good candidate susceptibility genes to this condition. The objectives of the present study were to determine if the 677T MTHFR and epsilon2/epsilon3/epsilon4 APOE alleles are risk factors for DM and for severity of diabetic retinopathy (DR). A total of 248 individuals were studied: 107 healthy individuals and 141 diabetic patients (46 with type 1 diabetes and 95 with type 2 diabetes), who also had DR (81 with non-proliferative DR and 60 with proliferative DR). The polymorphisms were analyzed by PCR followed by digestion with restriction enzyme or the single-nucleotide primer extension method. No evidence of association between the 677TT genotype of MTHFR gene and DM [cases: TT = 10/95 (10.6 percent); controls: TT = 14/107 (13 percent)] or with severity of DR was observed [cases: TT = 5/60 (8.5 percent); controls: TT = 9/81 (11.1 percent); P > 0.05]. We also did not find evidence of an association between APOE alleles and proliferative DR (epsilon2, epsilon3 and epsilon4 in cases: 9, 76, and 15 percent, and in controls: 5, 88, and 12 percent, respectively) but the carriers of epsilon2 allele were more frequent among patients with type 2 DM and DR than in controls [cases: 15/95 (15.8 percent); controls: 7/107 (6.5 percent); P < 0.05]. Therefore, our results suggest that the epsilon2 allele/APOE might be a risk factor for diabetes in the Brazilian population.