Rare crested rat subfossils unveil Afro-Eurasian ecological corridors synchronous with early human dispersals.

Biotic interactions between Africa and Eurasia across the Levant have invoked particular attention among scientists aiming to unravel early human dispersals. However, it remains unclear whether behavioral capacities enabled early modern humans to surpass the Saharo-Arabian deserts or if climatic changes triggered punctuated dispersals out of Africa. Here, we report an unusual subfossil assemblage discovered in a Judean Desert's cliff cave near the Dead Sea and dated to between ∼42,000 and at least 103,000 y ago. Paleogenomic and morphological comparisons indicate that the specimens belong to an extinct subspecies of the eastern African crested rat, Lophiomys imhausi maremortum subspecies nova, which diverged from the modern eastern African populations in the late Middle Pleistocene ∼226,000 to 165,000 y ago. The reported paleomitogenome is the oldest so far in the Levant, opening the door for future paleoDNA analyses in the region. Species distribution modeling points to the presence of continuous habitat corridors connecting eastern Africa with the Levant during the Last Interglacial ∼129,000 to 116,000 y ago, providing further evidence of the northern ingression of African biomes into Eurasia and reinforcing previous suggestions of the critical role of climate change in Late Pleistocene intercontinental biogeography. Furthermore, our study complements other paleoenvironmental proxies with local-instead of interregional-paleoenvironmental data, opening an unprecedented window into the Dead Sea rift paleolandscape.

Automated, high-throughput derivation, characterization and differentiation of induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are an essential tool for modeling how causal genetic variants impact cellular function in disease, as well as an emerging source of tissue for regenerative medicine. The preparation of somatic cells, their reprogramming and the subsequent verification of iPSC pluripotency are laborious, manual processes limiting the scale and reproducibility of this technology. Here we describe a modular, robotic platform for iPSC reprogramming enabling automated, high-throughput conversion of skin biopsies into iPSCs and differentiated cells with minimal manual intervention. We demonstrate that automated reprogramming and the pooled selection of polyclonal pluripotent cells results in high-quality, stable iPSCs. These lines display less line-to-line variation than either manually produced lines or lines produced through automation followed by single-colony subcloning. The robotic platform we describe will enable the application of iPSCs to population-scale biomedical problems including the study of complex genetic diseases and the development of personalized medicines.

Improved methods for reprogramming human dermal fibroblasts using fluorescence activated cell sorting.

Current methods to derive induced pluripotent stem cell (iPSC) lines from human dermal fibroblasts by viral infection rely on expensive and lengthy protocols. One major factor contributing to the time required to derive lines is the ability of researchers to identify fully reprogrammed unique candidate clones from a mixed cell population containing transformed or partially reprogrammed cells and fibroblasts at an early time point post infection. Failure to select high quality colonies early in the derivation process results in cell lines that require increased maintenance and unreliable experimental outcomes. Here, we describe an improved method for the derivation of iPSC lines using fluorescence activated cell sorting (FACS) to isolate single cells expressing the cell surface marker signature CD13(NEG)SSEA4(POS)Tra-1-60(POS) on day 7-10 after infection. This technique prospectively isolates fully reprogrammed iPSCs, and depletes both parental and "contaminating" partially reprogrammed fibroblasts, thereby substantially reducing the time and reagents required to generate iPSC lines without the use of defined small molecule cocktails. FACS derived iPSC lines express common markers of pluripotency, and possess spontaneous differentiation potential in vitro and in vivo. To demonstrate the suitability of FACS for high-throughput iPSC generation, we derived 228 individual iPSC lines using either integrating (retroviral) or non- integrating (Sendai virus) reprogramming vectors and performed extensive characterization on a subset of those lines. The iPSC lines used in this study were derived from 76 unique samples from a variety of tissue sources, including fresh or frozen fibroblasts generated from biopsies harvested from healthy or disease patients.

Nuclear genome transfer in human oocytes eliminates mitochondrial DNA variants.

Mitochondrial DNA mutations transmitted maternally within the oocyte cytoplasm often cause life-threatening disorders. Here we explore the use of nuclear genome transfer between unfertilized oocytes of two donors to prevent the transmission of mitochondrial mutations. Nuclear genome transfer did not reduce developmental efficiency to the blastocyst stage, and genome integrity was maintained provided that spontaneous oocyte activation was avoided through the transfer of incompletely assembled spindle-chromosome complexes. Mitochondrial DNA transferred with the nuclear genome was initially detected at levels below 1%, decreasing in blastocysts and stem-cell lines to undetectable levels, and remained undetectable after passaging for more than one year, clonal expansion, differentiation into neurons, cardiomyocytes or ß-cells, and after cellular reprogramming. Stem cells and differentiated cells had mitochondrial respiratory chain enzyme activities and oxygen consumption rates indistinguishable from controls. These results demonstrate the potential of nuclear genome transfer to prevent the transmission of mitochondrial disorders in humans.