Protocol for isolating human BCAM-positive corneal progenitor cells by flow cytometry and cell sorting.

BCAM-positive basal limbal epithelial cells are an early transit-amplifying cell population (TAC) capable of holoclone formation and corneal epithelial differentiation. Here, we present a protocol for isolating BCAM-positive cells from human donor corneas by flow cytometry and cell sorting. We describe steps for cell dissection and dissociation, antibody staining, and flow cytometry. We then detail procedures for culturing the purified BCAM-positive and BCAM-negative cells for holoclone and cell sheet formation assays to study the factors that regulate corneal regeneration. For complete details on the use and execution of this protocol, please refer to Sasamoto et al.1.

Genetic variation in ABCB5 associates with risk of hepatocellular carcinoma.

Expression of ATP-binding cassette B5 (ABCB5) has been demonstrated to confer chemoresistance, enhance cancer stem cell properties and associate with poor prognosis in hepatocellular carcinoma (HCC). The aim of this study was to evaluate the genetic variations of ABCB5 in HCC patients with reference to healthy individuals and the clinicopathological significance. A pilot study has examined 20 out of 300 pairs HCC and paralleled blood samples using conventional sequencing method to cover all exons and exon/intron regions to investigate whether there will be novel variant sequence and mutation event. A total of 300 HCC and 300 healthy blood DNA samples were then examined by Sequenom MassARRAY genotyping and pyrosequencing for 38 SNP and 1 INDEL in ABCB5. Five novel SNPs were identified in ABCB5. Comparison of DNA from blood samples of HCC and healthy demonstrated that ABCB5 SNPs rs75494098, rs4721940 and rs10254317 were associated with HCC risk. Specific ABCB5 variants were associated with aggressive HCC features. SNP rs17143212 was significantly associated with ABCB5 expression level. Nonetheless, the paralleled blood and tumour DNA sequences from HCC patients indicated that ABCB5 mutation in tumours was not common and corroborated the TCGA data sets. In conclusion, ABCB5 genetic variants had significant association with HCC risk and aggressive tumour properties.

Plasma DNA End-Motif Profiling as a Fragmentomic Marker in Cancer, Pregnancy, and Transplantation.

Plasma DNA fragmentomics is an emerging area of research covering plasma DNA sizes, end points, and nucleosome footprints. In the present study, we found a significant increase in the diversity of plasma DNA end motifs in patients with hepatocellular carcinoma (HCC). Compared with patients without HCC, patients with HCC showed a preferential pattern of 4-mer end motifs. In particular, the abundance of plasma DNA motif CCCA was much lower in patients with HCC than in subjects without HCC. The aberrant end motifs were also observed in patients with other cancer types, including colorectal cancer, lung cancer, nasopharyngeal carcinoma, and head and neck squamous cell carcinoma. We further observed that the profile of plasma DNA end motifs originating from the same organ, such as the liver, placenta, and hematopoietic cells, generally clustered together. The profile of end motifs may therefore serve as a class of biomarkers for liquid biopsy in oncology, noninvasive prenatal testing, and transplantation monitoring. SIGNIFICANCE: Plasma DNA molecules originating from the liver, HCC and other cancers, placenta, and hematopoietic cells each harbor a set of characteristic plasma DNA end motifs. Such markers carry tissue-of-origin information and represent a new class of biomarkers in the nascent field of fragmentomics.This article is highlighted in the In This Issue feature, p. 627.

Measuring fragmentation of ambulatory care in a tripartite healthcare system.

BACKGROUND: Hong Kong has a tripartite healthcare system, where western medicine provided in both public and private sectors coexist with Chinese medicine practice. The purpose of this study is to measure fragmentation of ambulatory care experienced by the non-institutionalized population aged 15 and over in such a tripartite system, thus shed light on the ongoing primary care reform. METHODS: This is a cross-sectional secondary data analysis using the Thematic Household Survey, which was conducted by the Hong Kong Census and Statistics Department during November 2009 to February 2010 to collect territory-wide health-related information. Among 18,226 individuals with two or more ambulatory visits during the past 12 months before interview, we grouped each visit into one of the three care segments-public western, private western and Chinese medicine. Two individual-level measures were used to quantify longitudinal fragmentation of care across segments over the one-year period: Most Frequent Provider Continuity Index (MFPC) and Fragmentation of Care Index (FCI). Both are analyzed for distribution and subgroup comparison. A Tobit model was used to further examine the determinants of fragmentation. RESULTS: More than a quarter of individuals sought care in two or all three segments, with an average MFPC of 65% and FCI of 0.528. Being older, female, married, unemployed, uninsured, or born in mainland China, with lower education, lower income, higher number of chronic conditions or poorer health were found to have experienced higher fragmentation of care. We also found that, fragmentation of care increased with the total number of ambulatory care visits and it varied significantly depending on what segment the individual chose to visit most frequently-those chose private western clinics had lower FCI, compared with those chose public western or Chinese medicine as the most frequently visited segment. CONCLUSIONS: Even measured at healthcare segment level, people in Hong Kong experienced modest fragmentation of care. Individuals' health beliefs-as a result of the persistent habitual tendency and latitude incentivized by the system-may be behind the fragmented care we saw. Efforts are needed to alter health beliefs, targeting subgroups of vulnerable population, and create environments that promote better coordinated primary care.

Presence of donor-derived DNA and cells in the urine of sex-mismatched hematopoietic stem cell transplant recipients: implication for the transrenal hypothesis.

BACKGROUND: The term "transrenal DNA" was coined in 2000 to signify that DNA in urine may come from the passage of plasma DNA through the kidney barrier. Although DNA in the urine has the potential to provide a completely noninvasive source of nucleic acids for molecular diagnosis, its existence remains controversial. METHODS: We obtained blood and urine samples from 22 hematopoietic stem cell transplant (HSCT) recipients and used fluorescence in situ hybridization, PCR for short tandem repeats, mass spectrometry, quantitative PCR, and immunofluorescence detection to study donor-derived DNA in the urine. RESULTS: All HSCT recipients exhibited high amounts of donor-derived DNA in buffy coat and plasma samples. Male donor-derived DNA was detected in supernatants of urine samples from all 5 female sex-mismatched HSCT recipients. Surprisingly, the amount of DNA in urine supernatants was not correlated with the plasma value. Moreover, cell-free urine supernatants contained DNA fragments >350 bp that were absent in plasma. Donor-derived polymorphs were detected in urine by fluorescence in situ hybridization. Coincidentally, donor-derived cytokeratin-producing epithelial cells were discovered in urine samples from 3 of 10 sex-mismatched HSCT recipients as long as 14.2 years after transplantation. CONCLUSIONS: This report is the first to demonstrate the presence of donor-derived DNA in the urine of HSCT recipients; however, we show that much of this DNA originates from donor-derived cells, rather than from the transrenal passage of cell-free plasma DNA. Our discovery of donor-derived cytokeratin-producing epithelial cells raises interesting biological and therapeutic implications, e.g., the capacity of marrow stem cells to serve as an extrarenal source for renal tubule regeneration.