Changes in CDKN2D , TP53, and miR125a expression: potential role in the evaluation of human amniotic fluid-derived mesenchymal stromal cell fitness.

Human amniotic fluid-derived mesenchymal stromal cells (hAMSC) have become one of the main cell populations used in regenerative medicine and for the study of various clinical disorders. These cells have a great capacity for proliferation and differentiation and do not form teratomas when transplanted into animal models, and their stemness seems to be between embryonic cells and adult mesenchymal cells. Before their use in cell therapy, they must be cultured and expanded in vitro, but the effect this process has on their fitness, a determining factor for the success or failure of cell therapy, is unknown. We undertook a follow-up of gene and microRNAs (miRNAs) expression using microarray of hAMSC for the first 15 passages. Significant changes were noted in the expression of various mRNAs and miRNAs, particularly down-regulation of TP53, increased expression of hsa-miR-125a and up-regulation of CDKN2D . The variations in TP53 and hsa-miR-125a may act as an indicator of the stemness of the hAMSC, whereas CDKN2D may indicate the begging of early senescence process in a p53-independent mechanism. The genes described in this study will help evaluate the fitness of hAMSC, thus guaranteeing their biological quality for use in regenerative medicine.

Evaluation of a low cost cryopreservation system on the biology of human amniotic fluid-derived mesenchymal stromal cells.

BACKGROUND: Human amniotic-derived mesenchymal stromal cells (hAMSC) are a novel population of multipotent stem cells that have been shown to have great potential for use in regenerative medicine. However, procedures to store and preserve hAMSC for future clinical applications have not been explored extensively. METHODS: In this study, we analyzed the influence of cryopreservation, using a protocol based on freezing rate of 1 °C/min, 10% dimethyl sulfoxide as cryoprotectant and a thawing rate >100 °C/min, on hAMSC morphology, proliferation rates, viability, cell cycle, karyotype, immune phenotype and multilineage differentiation potential. RESULTS: This study found that this cryopreservation protocol does not affect the biological properties of hAMSC. DISCUSSION: This shows that this protocol is a viable system for banking hAMSC, with the associated advantages that has a low cost in terms of expense, time and personnel involved and is easy to implement.

Lifespan of human amniotic fluid-derived multipotent mesenchymal stromal cells.

BACKGROUND AIMS: Human multipotent mesenchymal stromal cells (hMSC) have become one of the main interests in regenerative medicine because of their ability to differentiate into different lineages. Human amniotic fluid is reported to contain MSC (hAMSC) and therefore may be a useful source of cells for clinical applications. However, our understanding of the behavior of these cells in indefinite in vitro culture conditions is very limited. METHODS: We systematically evaluated and characterized, throughout their whole lifespan, the expansion potential, chromosomal stability, surface and intracellular phenotype and differentiation potential of fibroblastoid hAMSC (F-type hAMSC). RESULTS: Nine F-type hAMSC cultures could be expanded in in vitro culture conditions for 223.25 ± 24.44 days (mean ± SD), during which time 28.96 ± 1.5 passages were made giving rise to 54.95 ± 3.17 population doublings (PD) and an estimated number of accumulated cells of between 1.0 × 10(22) and 9.7 × 10(23), with no visible alterations in the chromosome during their lifespan. All the cultures showed unchanged percentages of strongly positive expressions of the surface markers CD29, CD44, CD73, CD90, CD95, CD105 and HLA-ABC, as well as the embryonic intracellular markers Nanog and Sox2, during their lifespan, whereas the expression of the embryonic surface markers SSEA3, SSEA4, TRA-1-60 and TRA-1-81 fell until it disappeared with progression of the culture. These cells retained their differentiation capacities to adipogenic, chondrogenic and osteogenic lineages throughout their lifespan. CONCLUSIONS: F-type hAMSC exhibit reproducible biologic characteristics, confirming that these cells are ideal candidates for use in regenerative medicine.

High prevalence of hepatitis C virus subtypes 4c and 4d in Malaga (Spain): phylogenetic and epidemiological analyses.

Hepatitis C virus (HCV) is a major aetiological agent of chronic hepatitis and it may lead to the development of liver cirrhosis and hepatocellular carcinoma. HCV has been classified into six clades as a result of high genetic variability. A commercial procedure to genotype HCV in 678 patients from Carlos Haya Regional University Hospital, Malaga was used to study the distribution of HCV genotypes in Malaga, southern Spain. A high prevalence of HCV-4 (10.2%) was found. This genotype is found more commonly in Egypt, Central Africa and the Middle East. The distribution of the different subtypes in the 69 patients with HCV-4 was as follows: 4.3% subtype 4e, 7.2% subtype 4a, 11.5% not subtypable, and 76.8% subtype 4c/4d. Of the 53 4c/4d patients, 69% were intravenous drug users and 31% non-intravenous drug users. In order to characterise further the HCV-4c/4d patients, sequences of the non-structural 5B gene (393 bp) were obtained from 36 HCV-4c/4d-infected untreated patients. Phylogenetic tree topologies distinguished clearly the two subtypes: 11 patients were infected by subtype 4c and 25 by 4d. This phylogenetic analysis, reinforced by the epidemiological characteristics, suggests the extension of the HCV-4c and -4d subtypes in the area of Malaga among both intravenous drug users and non-intravenous drug users.

Differential transcriptional expresión of the polymorphic myxovirus resistance protein A in response to interferon-alpha treatment.

Levels of myxovirus resistance protein A (MxA) mRNA were studied for a single nucleotide polymorphism in the promoter region at nucleotide position -88 of the gene to identify individual-specific responses to interferon (IFN)-alpha2 that might predict responsiveness to IFN-alpha therapy. We quantified MxA expression by reverse transcription and real-time polymerase chain reaction in peripheral blood mononuclear cells (PBMC) in vitro, induced by IFN-alpha2, from 22 healthy donors, in relation with G/T polymorphism located in the promoter of the MxA. MxA mRNA was significantly upregulated in all subjects (mean of 53-fold) in response to IFN-alpha2 in vitro (P < 0.01). Comparison of the inducibility of MxA mRNA expression in relation with G/T polymorphism showed a 4.26-fold higher induction of MxA mRNA levels in PBMC from carriers of the mutant allele (GT or TT) than homozygotes with the wild-type allele (GG) (P < 0.001). We propose that expression of the IFN-inducible MxA is affected by a single nucleotide polymorphism in the MxA promoter which can identify an individual response to IFN-alpha2.