Serum-converted platelet lysate can substitute for fetal bovine serum in human mesenchymal stromal cell cultures.

BACKGROUND AIMS: Fetal bovine serum (FBS) is commonly used as a serum supplement for culturing human mesenchymal stromal cells (hMSCs). However, human cells grown in FBS, especially for extended periods, risk potential exposure to bovine immunogenic proteins and infectious agents. To address this issue, we investigated the ability of a novel human platelet serum supplement to substitute for FBS in hMSC cultures. METHODS: Platelet lysate-serum (PL-serum) was converted from platelet lysate-plasma (PL-plasma) that was manufactured from pooled platelet-rich plasma (PRP) apheresis units. Growth factor levels and the number of residual intact platelets in PL-serum and PL-plasma were compared with enzyme-linked immunosorbent assays and flow cytometry, respectively. Proliferation responses of hMSCs cultured in PL-serum, PL-plasma, or FBS were assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the immunophenotype of harvested hMSCs was evaluated by flow cytometry and tri-lineage differentiation potential was evaluated by assessing adipogenic, osteogenic and chondrogenic development. RESULTS: Selected growth factor levels in PL-serum were not significantly different from PL-plasma (P > 0.05). hMSC cultures supplemented with PL-serum had comparable growth kinetics to PL-plasma, and hMSC yields were consistently greater than with FBS. hMSCs harvested from cultures supplemented with PL-serum, PL-plasma or FBS had similar cell surface phenotypes and maintained tri-lineage differentiation potential. CONCLUSIONS: PL-serum, similar to PL-plasma, can substitute for FBS in hMSC cultures. Use of PL-serum, in contrast to PL-plasma, has an added advantage of not requiring addition of a xenogeneic source of heparin, providing a completely xeno-free culture medium.

Advanced technology for storage and transport of purified DNA from umbilical cord blood prior to use in human leukocyte antigens typing and whole-genome microarray analysis.

Two technologies for dry-state, ambient temperature transport of biospecimens were evaluated in this study. Umbilical cord blood (UCB) samples from 4 individuals were transported at ambient temperature using GenPlates, and the DNA recovered was compared with DNA purified directly from granulocytes of the same UCB samples. GenTegra™ DNA tubes were then used to transport the DNA from California to North Carolina and New Zealand, either immediately after drying or following 30 days of storage at 25°C and 76°C. The integrity of the recovered DNA was thoroughly tested using 2 human leukocyte antigens (HLA)-typing techniques (bead array and sequencing), as well as microarray-based whole-genome scanning. HLA-typing results were the same for all samples whether the DNA had been stored for 3 days during transport or 30 days at either 25°C or 76°C. There were no differences in the HLA-typing results of DNA recovered from UCB samples stored in GenPlates compared with DNA extracted directly from granulocytes. Moreover, the microarray analysis revealed call rates of >99.5% for every sample, regardless of storage method, with a statistical concordance of 99.99% between the UCB samples stored in GenPlates compared with DNA extracted directly from granulocytes. These results indicate that both GenPlates and GenTegra are viable methods of storing and transporting UCB (stem cell) biospecimens in a dry state. The quality and quantity of DNA recovered using both technologies are sufficient for complex genotyping using a number of different methods.

Missense mutation of the last nucleotide of exon 1 (G->C) of beta globin gene not only leads to undetectable mutant peptide and transcript but also interferes with the expression of wild allele.

Hemoglobin Monroe (beta globin G->C, codon 30) is a missense mutation. We could not detect either the mutant peptide or transcript in reticulocyte-enriched preparation and in expanded erythroid progenitor cells. By quantitative gene expression assay beta globin mRNA was found to be reduced by more than 70% in all heterozygous subjects with different haplotypes. We conclude that this mutation also interferes with expression of wild type allele.

Familial polycythemia caused by a novel mutation in the beta globin gene: essential role of P50 in evaluation of familial polycythemia.

Two polycythemic subjects from a family with multiple polycythemic subjects were evaluated. Estimation of oxygen affinity of Hb from venous blood gas parameters (P50) revealed low P50 suggesting a high affinity Hb variant. Further work up, which included beta globin gene sequencing, revealed a novel mutation changing a codon to the previously reported high affinity Hb - Hb Johnstown (beta 109 Val->Leu). Polycythemic subjects with high affinity Hb variant are asymptomatic with normal life expectancy. Their differentiation from polycythemia vera (PV) is crucial to avoid therapy which is otherwise reserved for PV patients. We provide an electronic version (in Microsoft excel program) of a previously reported mathematical formula for rapid calculation of P50 from venous blood gases. Estimation of P50 is an essential initial step in the evaluation of a subject with personal and family history of polycythemia.

Possible immunomodulatory actions of Carica papaya seed extract.

Carica papaya seed extract is currently being marketed as a nutritional supplement with purported ability "to rejuvenate the body condition and to increase energy". The product claims to improve immunity against common infection and body functioning. The present study was initiated to analyze the chemical constituents of the Carica Seed Extract and determine the potential immunomodulatory properties of the different bioactive fractions. These immunomodulatory activities of crude Carica Seed Extract and its bioactive fractions were examined in vitro using lymphocyte proliferation assays and complement-mediated hemolytic assay. Three major observations were made in this study: (1) the crude Carica Seed Extract and two other bioactive fractions significantly enhanced the phytohemagglutinin responsiveness of lymphocytes; (2) none of the Carica Seed Extract (at the concentrations used in this study) was able to protect the lymphocytes from the toxic effects of chromium; and (3) some of the bioactive fractions of Carica Seed Extract were able to significantly inhibit the classical complement-mediated hemolytic pathway. These findings provide evidence for immunostimulatory and anti-inflammatory actions of Carica Seed Extract. No single compound is likely responsible for these activities. Further purification, isolation and characterization of the active components are needed.