Biology subjects

Kaufman, D. S.

Publications and source records attributed to Kaufman, D. S..

9 recordsLinked to original sources

bioRxiv2025

Derivation of functional early gestation decidual natural killer cell subtypes from induced pluripotent stem cells

Abnormal decidual natural killer cell (dNK) function is linked to pregnancy complications occurring in both early and late gestation, including recurrent pregnancy loss, preeclampsia, and preterm birth. Exploration of dNK heterogeneity as it relates to function is an active area of research; however, most of this work has focused on early gestation. Using flow cytometric and transcriptomic single-cell definitions of dNK subtypes, we characterized dNK heterogeneity in term dNK within both chorioamniotic membranes and basal plate. We also applied aptamer-based secretome profiling to first trimester and term dNK, and found dNK-specific proteins - VEGF and PLGF - to be reduced at term. We further determined that, compared to first trimester dNK, term dNK have reduced cytoxicity against target cells. Finally, we applied this knowledge to establish a protocol for differentiation of induced pluripotent stem cells (iPSC) into functional dNK. We found that treatment with TGF{beta} enriched for dNK2 subtype, while inducing dNK markers, CD9 and CD103. We evaluated function using cytokine and degranulation assays, aptamer-based secretome profiling, and cytotoxicity assays. We found that iPSC-dNK are functionally most similar to primary dNK. Further, TGF{beta} iPSC-dNK had reduced GM-CSF in response to PMA/I and increased secretion of VEGF and other first trimester-specific proteins - supportive of a shift towards an early gestation, dNK2-dominant, phenotype. We conclude that changes in dNK function across gestation reflect shifts in dNK subtypes that can be reproducibly derived from iPSC, providing a new method for modeling dNK and laying the foundation for cell-based therapeutics for reproductive disease. Significance StatementAlterations in maternal decidual natural killer cells (dNK) are associated with pregnancy complications - from recurrent pregnancy loss to preeclampsia and preterm birth. We found dNK from different regions of the term placenta to be distinct from peripheral blood NK and early gestation dNK, based on gene and surface marker expression, subtype composition, secretome, and cytotoxicity. We report a novel, reproducible protocol to generate dNK resembling the most abundant dNK subtype in early gestation from induced pluripotent stem cells. Our study lays the foundation for in vitro modeling of the maternal-fetal interface and therapeutic development for reproductive disease.

cell biology↗

bioRxiv2025

Dynamic connectivities of plant metacommunities at a millennial time-scale: the Beringia testbed

Ecological connectivity shapes ecosystem responses to climate change and is thought to underpin stability, yet its millennial-scale dynamics remain poorly resolved. We asked how spatial and temporal connectivity of plant metacommunities changed over the last 40 ka and which processes drove it. We analysed and compiled plant sedimentary DNA from 20 lake cores across Beringia (Siberia, Alaska) to investigate community dynamics and, for a high-resolution subset, applied beta- and zeta-diversity to track connectivity. Vegetation changed coherently across the glacial-Holocene transition, with trait shifts mirroring functional composition. Connectivity peaked during the late MIS3 and Last Glacial Maximum--likely aided by the Bering Land Bridge, mass effects and facilitation--collapsed during the Deglacial with rapid turnover, and rebounded in the Holocene as shrub and boreal communities expanded. Temporal zeta within sites exceeded spatial zeta, indicating strong local persistence and resilience. Tundra sites uninvaded by forest maintained continuous species pools. Overall, these patterns underscore the value of a metacommunity perspective for assessing millennial-scale connectivity changes.

ecology↗

bioRxiv2025

Toggling of NKG2A expression drives functional specialization of iPSC-derived CAR NK cells

Induced pluripotent stem cell (iPSC)-derived natural killer (iNK) cells offer a promising platform for off-the-shelf immunotherapy against hematological malignancies. NK cell function is dynamically regulated through education driven by inhibitory receptors, including CD94/NKG2A and killer cell immunoglobulin-like receptors (KIR). However, the acquisition of inhibitory receptors in iNK cells and their role during differentiation and education remains poorly defined. In this study, we monitored receptor repertoires, transcriptional states, and functional responses in a range of genetically engineered iNK cell lines. Transcriptional reference mapping placed iNK cells close to cytokine-activated NKG2A+ CD56dim peripheral blood (PB) NK cells. Despite their early differentiation stage, iNK cells displayed a well-developed cytotoxic effector program, which was also reflected in high protein expression of Eomes, granzyme B, and activating receptors DNAM-1 and NKG2D. Acquisition of NKG2A by iNK cells was associated with a more differentiated transcriptional state and superior functional responses against a broad range of targets, including those expressing low to moderate levels of HLA-E, suggesting attenuated inhibitory signaling through NKG2A in iNKs. CRISPR knockout of {beta}2-microglobulin (B2) in iNK cells revealed that the functional potency of NKG2A+ iNK cells was independent of educating interactions with HLA-E in cis or trans. Finally, CRISPR-mediated ablation of NKG2A led to a spontaneous compensatory surface expression of CD94/NKG2C heterodimers, associated with enhanced IFN-{gamma} production and cytotoxic activity against target cells with forced high expression of single-chain {beta}2m-HLA-E-peptide trimers. Our results indicate an education-independent functional maturation of iNK cells, characterized by potent effector programs coupled with a favorable early-stage transcriptional profile.

immunology↗

bioRxiv2025

Arctic defaunation initiated a cascade of mammal-plant interaction shift through dispersal dynamics

Amid accelerating defaunation, it remains unclear whether the loss of large mammals can trigger ecosystem collapse and through which functional pathways. Using sedimentary ancient metagenomics, we identify an overlooked role of large mammal defaunation in helping drive the collapse of the glacial mammoth-steppe. Network analyses identified rewiring of mammal interactions during the late glacial supporting cascading effects. Crucially, megafaunal-mediated long-distance seed dispersal buffered vegetation turnover during periods of rapid climate warming but weakened with progressive losses of vital dispersal interactions leading to the steppe-tundras transition to open woodland in the early Holocene. Trophic interactions did not offset turnover and became dominant afterwards with the more stable climate. We identified resilient interactions that may support recovery, suggesting the potential of rewilding to restore Arctic ecosystems. One-sentence abstractAncient metagenomics reveals disrupted long-distance seed dispersal helped drive mammoth-steppe collapse and informs Arctic rewilding efforts.

ecology↗

bioRxiv2025

Unveiling Genomic Rearrangements in Engineered iPSC Lines by Optical Genome Mapping

We demonstrate here the use of optical genome mapping (OGM) to detect genetic alterations arising from gene editing by various technologies in human induced pluripotent stem cells (iPSCs). OGM enables an unbiased and comprehensive analysis of the entire genome, allowing the detection of genomic structural variants (SVs) of all classes with a quantitative variant allele frequency (VAF) sensitivity of 5%. In this pilot study, we conducted a comparative dual analysis between the parental iPSCs and the derived cells that had undergone gene editing using various techniques, including transposons, lentiviral transduction, and CRISPR-Cas9-mediated safe harbor locus insertion at the adeno-associated virus integration site 1 (AAVS1). These analyses demonstrated that iPSCs that had been edited using transposons or lentiviral transduction resulted in a high number of transgene insertions in the genome. In contrast, CRISPR-Cas9 technology resulted in a more precise and limited transgene insertion, with only a single target sequence observed at the intended locus. These studies demonstrate the value of OGM to detect genetic alterations in engineered cell products and suggests that OGM, together with DNA sequencing, could be a valuable tool when evaluating genetically modified iPSCs for research and therapeutic purposes.

molecular biology↗

bioRxiv2025

Integrative CRISPR Screens and RNA-Omics Discover an Essential Role for PUF60-3' Splice Site Interactions in Cancer Progression

RNA-binding proteins (RBPs) are important regulators of post-transcriptional gene expression. Understanding which and how RBPs promote cancer progression is crucial for cancers that lack effective targeted therapies such as triple negative breast cancer (TNBC). Here, we employ both in vitro and in vivo pooled CRISPR/Cas9 screening to identify 50 RBP candidates that are essential for TNBC cell survival. Integrated eCLIP and RNA-sequencing analysis identify that poly(U)-binding splicing factor 60 (PUF60) drives exon inclusion within proliferation-associated transcripts that, when mis-spliced, induce cell cycle arrest and DNA damage. Furthermore, disrupting PUF60 interactions with 3 splice sites via a substitution in its RNA-binding domain causes widespread exon skipping, leading to downregulation of proliferation-associated mRNAs and inducing apoptosis in TNBC cells. We demonstrate that loss of PUF60-RNA interactions inhibits TNBC cell proliferation and shrinks tumor xenografts, revealing the molecular mechanism by which PUF60 supports cancer progression. SignificanceOur work demonstrates functional in vivo screening of RBPs as an effective strategy for identifying unexpected cancer regulators. Here, we reveal a crucial role for PUF60-mediated splicing activity in supporting oncogenic proliferation rates and highlight its potential as a therapeutic target in triple negative breast cancer.

cancer biology↗

bioRxiv2024

XOL-1 regulates developmental timing by modulating the H3K9 landscape in C. elegans early embryos

Sex determination in the nematode C. elegans is controlled by the master regulator XOL-1 during embryogenesis. Expression of xol-1 is dependent on the ratio of X chromosomes and autosomes, which differs between XX hermaphrodites and XO males. In males, xol-1 is highly expressed and in hermaphrodites, xol-1 is expressed at very low levels. XOL-1 activity is known to be critical for the proper development of C. elegans males, but its low expression was considered to be of minimal importance in the development of hermaphrodite embryos. Our study reveals that XOL-1 plays an important role as a regulator of developmental timing during hermaphrodite embryogenesis. Using a combination of imaging and bioinformatics techniques, we found that hermaphrodite embryos have an accelerated rate of cell division, as well as a more developmentally advanced transcriptional program when xol-1 is lost. Further analyses reveal that XOL-1 is responsible for regulating the timing of initiation of dosage compensation on the X chromosomes, and the appropriate expression of sex-biased transcriptional programs in hermaphrodites. We found that xol-1 mutant embryos overexpress the H3K9 methyltransferase MET-2 and have an altered H3K9me landscape. Some of these effects of the loss of xol-1 gene were reversed by the loss of met-2. These findings demonstrate that XOL-1 plays an important role as a developmental regulator in embryos of both sexes, and that MET-2 acts as a downstream effector of XOL-1 activity in hermaphrodites. Author SummaryVarious organisms have differing ways of determining, at a molecular level, what the sex of a developing embryo is supposed to be. The two sexes in the nematode C. elegans, hermaphrodite and male, have different numbers of X chromosomes. Hermaphrodites have two X chromosomes and males only have one. This mismatch raises an additional problem as hermaphrodites will have twice the amount of genes expressed from the X compared to males. This is solved by a process called dosage compensation, which equalizes gene expression between the sexes. A molecular sensor called XOL-1 detects the number of X chromosomes in an embryo and kick-starts the process of proper sexual development and/or dosage compensation. XOL-1 was known to be very important for activating male development but was believed to not have any role in the development of the hermaphrodite sex. We show that XOL-1 has some crucial roles in hermaphrodite embryos in controlling the rate of development of the embryo, and regulating the timing of dosage compensation. We also show that MET-2, a protein that deposits repressive methyl marks on DNA-bound histone proteins, is involved in this process.

genetics↗

bioRxiv2021

Transcriptional and Functional Activity of Canine Hemangiosarcoma to Support Hematopoiesis Demonstrate Bone Marrow Nurse Cell Ontogeny

Hemangiosarcoma and angiosarcoma are soft-tissue sarcomas of blood vessel-forming cells in dogs and humans, respectively. These vasoformative sarcomas are aggressive and highly metastatic, with disorganized, irregular blood-filled vascular spaces. Our objective was to define molecular programs which support the niche that enables progression of canine hemangiosarcoma and human angiosarcoma. Dog-in-mouse hemangiosarcoma xenografts recapitulated the vasoformative and highly angiogenic morphology and molecular characteristics of primary tumors. Blood vessels in the tumors were complex and disorganized, and they were lined by both donor and host cells, a trait that was not observed in xenografts from canine osteosarcoma and lymphoma. In some cases, the xenografted hemangiosarcoma cells created exuberant myeloid hyperplasia and gave rise to lymphoproliferative tumors of mouse origin. We did not uncover a definitive transmissible etiology, but our functional analyses indicate that hemangiosarcoma cells generate a microenvironment that supports expansion and differentiation of hematopoietic progenitor populations. We conclude that canine hemangiosarcomas, and possibly human angiosarcomas, originate from stromal cells that are part of the bone marrow niche and that these cells may also support the growth of hematopoietic tumors. SignificanceWe demonstrate that molecular programs supporting expansion of immune and inflammatory cells in hemangiosarcoma resemble those of bone marrow niche cells, providing insights into the potential roles of these cells - whether physiological or pathological - in creating a permissive environment for the progression of hematopoietic malignancies.

cancer biology↗

bioRxiv2019

An improved method to produce clinical scale natural killer cells from human pluripotent stem cells

Human natural killer (NK) cell-based adoptive anti-cancer immunotherapy has gained intense interest with many clinical trials actively recruiting patients to treat a variety of both hematological malignancies and solid tumors. Most of these trials use primary NK cells isolated either from peripheral blood (PB-NK cells) or umbilical cord blood (UCB-NK cells), though these sources require NK cell collection for each patient leading to donor variability and heterogeneity in the NK cell populations. In contrast, NK cells derived human embryonic stem cells (hESC-NK cells) or induced pluripotent stem cells (hiPSC-NK cells) provide more homogeneous cell populations that can be grown at clinical scale, and genetically engineered if desired. These characteristics make hESC/iPSC-derived NK cells an ideal cell population for developing standardized, \"off-the-shelf\" immunotherapy products. Additionally, production of NK cells from undifferentiated human pluripotent stem cells enables studies to better define pathways that regulate human NK cell development and function. Our group previously established a stromal-free, two-stage culture system to derive NK cells from hESC/hiPSC in vitro followed by clinical-scale expansion of these cells using interleukin-21 expressing artificial antigen-presenting cells. However, prior to differentiation, this method requires single cell adaption of hESCs/hiPSCs which takes months. Recently we optimized this method by adapting the mouse embryonic fibroblast-dependent hESC/hiPSC to feeder-free culture conditions. These feeder-free hESC/hiPSCs are directly used to generate hemato-endothelial precursor cells. This new method produces mature, functional NK cells with higher efficiency to enable rapid production of an essentially unlimited number of homogenous NK cells that can be used for standardized, targeted immunotherapy for the treatment of refractory cancers and infectious diseases.

immunology↗