Biology subjects

Ludmann, S. A.

Publications and source records attributed to Ludmann, S. A..

3 recordsLinked to original sources

bioRxiv2026

Multi-omic profiling of human antibody-secreting cells reveals diverse subsets sustain durable humoral immunity

Antibody-secreting cells (ASCs) provide humoral immunity that can mediate lifelong protection against pathogens. Current classifications cannot delineate the heterogenous functionalities, tissue residencies, and lifespans of human ASC subsets, impeding clinical translation. We applied multi-omic sequencing, spatial proteomics, and functional assays to discover and characterize human bone marrow (BM) ASC subsets. We identified two peripheral subsets (ASCp) also present in blood and three BM-resident subsets (ASCr), comprising a maturation continuum associated with increased mitochondrial networking, diminished antibody secretion, differential transcription factor motif accessibility, and preferential co-localization in homotypic niches. CD19+9+ASCr and CD19-ASCr exhibited poor recovery years after BM transplantation, indicating a strong dependence on supportive niches. Childhood vaccine antigens were recognized by long-lived ASCr subsets in adults and by immature HLA-DR+ASCp, implying ASCs can differentiate without recent antigen exposure. Our results provide new insights into ASC identity, maturation, and longevity and a generalizable framework for study and manipulation of human ASCs.

immunology↗

bioRxiv2018

Scarless gene tagging of transcriptionally silent genes in hiPSCs to visualize cardiomyocyte sarcomeres in live cells

We describe a multi-step CRISPR/Cas9 gene editing method to create endogenously tagged GFP-fusions of transcriptionally silent genes in human induced pluripotent stem cells (hiPSCs), allowing visualization of proteins that are only expressed upon differentiation. To do this, we designed a donor template containing the monomeric EGFP (mEGFP) fusion tag and an mCherry selection cassette delivered in tandem to a target locus via homology directed repair (HDR). mCherry expression was driven by a constitutive promoter and served as a drug-free, excisable selection marker. Following selection, the mCherry cassette was excised with Cas9, creating an mEGFP-fusion with the target gene. We achieved scarless excision by using repetitive sequences to guide microhomology-mediated end joining (MMEJ) and introduce linker sequences between the mEGFP tag and the target gene. Using this strategy, we successfully tagged genes encoding the cardiomyocyte sarcomeric proteins troponin I (TNNI1), alpha-actinin (ACTN2), titin (TTN), myosin light chain 2a (MYL7), and myosin light chain 2v (MYL2) with mEGFP in undifferentiated hiPSCs. This methodology provides a general strategy for scarlessly introducing tags to transcriptionally silent loci in hiPSCs.

cell biology↗

bioRxiv2017

Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization

We present a CRISPR/Cas9 genome editing strategy to systematically tag endogenous proteins with fluorescent tags in human inducible pluripotent stem cells. To date we have generated multiple human iPSC lines with GFP tags for 10 proteins representing key cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, lamin B1, non-muscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome editing methodology using Cas9 ribonuclear protein electroporation and fluorescence-based enrichment of edited cells resulted in <0.1-24% HDR across all experiments. Clones were generated from each edited population and screened for precise editing. [~]25% of the clones contained precise mono-allelic edits at the targeted locus. Furthermore, 92% (36/39) of expanded clonal lines satisfied key quality control criteria including genomic stability, appropriate expression and localization of the tagged protein, and pluripotency. Final clonal lines corresponding to each of the 10 cellular structures are now available to the research community. The data described here, including our editing protocol, genetic screening, quality control assays, and imaging observations, can serve as an initial resource for genome editing in cell biology and stem cell research.

cell biology↗