Reliability of phenotype estimation and extended classification of ancestry using decedent samples.

The Illumina® MiSeq FGx™, in conjunction with the ForenSeq™ DNA Signature Prep kit, produces genotypes of the CODIS-required short tandem repeats and provides phenotype and biogeographical ancestry estimations via phenotype-informative and ancestry-informative markers, respectively. Although both markers have been validated for use in forensic biology, there is little data to determine the practical utility of these estimations to assist in identifying missing persons using decedent casework samples. The accuracy and utility of phenotypic and ancestral estimations were investigated for 300 samples received by the Los Angeles County Department of Medical Examiner-Coroner. piSNP genotypes were translated into hair and eye colors using the Forenseq™ Universal Analysis Software (UAS) on the MiSeq FGx™ and the HIrisPlex System, and statistical accuracy was evaluated in context with the reported decedent characteristics. Similarly, estimates of each decedent's biogeographical ancestry were compared to assess the efficacy of these markers to predict ancestry correctly. The average UAS and the HIrisPlex system prediction accuracy for brown and blue eyes were 95.3% and 96.2%, respectively. Intermediate eye color could not be predicted with high accuracy using either system. Other than the black hair phenotype reporting an accuracy that exceeded 90% using either system, hair color was also too variable to be predicted with high accuracy. The FROG-kb database distinguishes decedents adequately beyond the Asian, African, European, and Admixed American global ancestries provided by the MiSeq FGx™ UAS PCA plots. FROG-kb correctly identified Middle Eastern, Pacific Islander, Latin American, or Jewish ancestries with accuracies of 70.0%, 81.8%, 73.8%, and 86.7%, respectively.

DNA methylation of decedent blood samples to estimate the chronological age of human remains.

Chronological age estimation may offer valuable investigative leads in human identification cases. Bisulfite pyrosequencing analysis of single CpG sites on five genes (KLF14, ELOVL2, C1orf132, TRIM59, and FHL2) was performed on 264 postmortem blood samples from individuals aged 3 months to 93 years. The goals were to develop age prediction models based on the correlation between the methylation profile and chronological age and to assess the accuracy of the prediction. Linear regression between methylation levels and age at each CpG site revealed that the five markers show a statistically significant correlation with age. The methylation data from a training set of 160 postmortem blood samples were used to develop an age prediction model with a correlation coefficient of 0.65, explaining 73.1% of age variation, with a mean absolute deviation from the chronological age of 7.60 years. The accuracy of the model was evaluated with a test set of 72 samples producing a mean absolute deviation of 7.42 years. The training and test sets were also categorized by specific age groups to assess accuracy and deviation from chronological age. The data for both sets revealed a lower prediction potential as an individual increases in age, particularly for the age categories above 50 years.

CD10 expression identifies a subset of human perivascular progenitor cells with high proliferation and calcification potentials.

The tunica adventitia ensheathes arteries and veins and contains presumptive mesenchymal stem cells (MSCs) involved in vascular remodeling. We show here that a subset of human adventitial cells express the CD10/CALLA cell surface metalloprotease. Both CD10+ and CD10- adventitial cells displayed phenotypic features of MSCs when expanded in culture. However, CD10+ adventitial cells exhibited higher proliferation, clonogenic and osteogenic potentials in comparison to their CD10- counterparts. CD10+ adventitial cells increased expression of the cell cycle protein CCND2 via ERK1/2 signaling and osteoblastogenic gene expression via NF-κB signaling. CD10 expression was upregulated in adventitial cells through sonic hedgehog-mediated GLI1 signaling. These results suggest that CD10, which marks rapidly dividing cells in other normal and malignant cell lineages, plays a role in perivascular MSC function and cell fate specification. These findings also point to a role for CD10+ perivascular cells in vascular remodeling and calcification.

Transcriptional Networks in Single Perivascular Cells Sorted from Human Adipose Tissue Reveal a Hierarchy of Mesenchymal Stem Cells.

Adipose tissue is a rich source of multipotent mesenchymal stem-like cells, located in the perivascular niche. Based on their surface markers, these have been assigned to two main categories: CD31- /CD45- /CD34+ /CD146- cells (adventitial stromal/stem cells [ASCs]) and CD31- /CD45- /CD34- /CD146+ cells (pericytes [PCs]). These populations display heterogeneity of unknown significance. We hypothesized that aldehyde dehydrogenase (ALDH) activity, a functional marker of primitivity, could help to better define ASC and PC subclasses. To this end, the stromal vascular fraction from a human lipoaspirate was simultaneously stained with fluorescent antibodies to CD31, CD45, CD34, and CD146 antigens and the ALDH substrate Aldefluor, then sorted by fluorescence-activated cell sorting. Individual ASCs (n = 67) and PCs (n = 73) selected from the extremities of the ALDH-staining spectrum were transcriptionally profiled by Fluidigm single-cell quantitative polymerase chain reaction for a predefined set (n = 429) of marker genes. To these single-cell data, we applied differential expression and principal component and clustering analysis, as well as an original gene coexpression network reconstruction algorithm. Despite the stochasticity at the single-cell level, covariation of gene expression analysis yielded multiple network connectivity parameters suggesting that these perivascular progenitor cell subclasses possess the following order of maturity: (a) ALDHbr ASC (most primitive); (b) ALDHdim ASC; (c) ALDHbr PC; (d) ALDHdim PC (least primitive). This order was independently supported by specific combinations of class-specific expressed genes and further confirmed by the analysis of associated signaling pathways. In conclusion, single-cell transcriptional analysis of four populations isolated from fat by surface markers and enzyme activity suggests a developmental hierarchy among perivascular mesenchymal stem cells supported by markers and coexpression networks. Stem Cells 2017;35:1273-1289.