Identification and characterization of in vitro expanded hematopoietic stem cells.

Hematopoietic stem cells (HSCs) cultured outside the body are the fundamental component of a wide range of cellular and gene therapies. Recent efforts have achieved > 200-fold expansion of functional HSCs, but their molecular characterization has not been possible since the majority of cells are non-HSCs and single cell-initiated cultures have substantial clone-to-clone variability. Using the Fgd5 reporter mouse in combination with the EPCR surface marker, we report exclusive identification of HSCs from non-HSCs in expansion cultures. By directly linking single-clone functional transplantation data with single-clone gene expression profiling, we show that the molecular profile of expanded HSCs is similar to proliferating fetal HSCs and reveals a gene expression signature, including Esam, Prdm16, Fstl1, and Palld, that can identify functional HSCs from multiple cellular states. This "repopulation signature" (RepopSig) also enriches for HSCs in human datasets. Together, these findings demonstrate the power of integrating functional and molecular datasets to better derive meaningful gene signatures and opens the opportunity for a wide range of functional screening and molecular experiments previously not possible due to limited HSC numbers.

Short-term behavioral and histological findings following a single concussive and repeated subconcussive brain injury in a rodent model.

PRIMARY OBJECTIVE: It is unclear of the correlation between a mild traumatic brain injury (mTBI) and repeated subconcussive (RSC) impacts with respect to injury biomechanics. Thus, the present study was designed to determine the behavioral and histological differences between a single mTBI impact and RSC impacts with subdivided cumulative kinetic energies of the single mTBI impact. RESEARCH DESIGN: Adult male Sprague-Dawley rats were randomly assigned to a single mTBI impact, RSC impact, sham, or repeated sham groups. METHODS AND PROCEDURES: Following a weight drop injury, anxiety-like behavior and general locomotive activity and were assessed using the open field test, while motor coordination was evaluated using a rotarod unit. Neuronal loss, astrogliosis, and microgliosis were assessed using NeuN, GFAP and Iba-1 immunohistochemistry. All assessments were undertaken at 3- and 7-days post impact. MAIN OUTCOMES AND RESULTS: No behavioral disturbances were observed in injury groups, however, both injury groups did lead to microgliosis following 3-days post-impact. CONCLUSIONS: No pathophysiological differences were observed between a single mTBI impact and RSC impacts of the same energy input. Even though a cumulative injury threshold for RSC impacts was not determined, a threshold still may exist where no pathodynamic shift occurs.

Convergent somatic evolution commences in utero in a germline ribosomopathy.

Clonal tracking of cells using somatic mutations permits exploration of clonal dynamics in human disease. Here, we perform whole genome sequencing of 323 haematopoietic colonies from 10 individuals with the inherited ribosomopathy Shwachman-Diamond syndrome to reconstruct haematopoietic phylogenies. In ~30% of colonies, we identify mutually exclusive mutations in TP53, EIF6, RPL5, RPL22, PRPF8, plus chromosome 7 and 15 aberrations that increase SBDS and EFL1 gene dosage, respectively. Target gene mutations commence in utero, resulting in a profusion of clonal expansions, with only a few haematopoietic stem cell lineages (mean 8, range 1-24) contributing ~50% of haematopoietic colonies across 8 individuals (range 4-100% clonality) by young adulthood. Rapid clonal expansion during disease transformation is associated with biallelic TP53 mutations and increased mutation burden. Our study highlights how convergent somatic mutation of the p53-dependent nucleolar surveillance pathway offsets the deleterious effects of germline ribosomopathy but increases opportunity for TP53-mutated cancer evolution.