Journal: bioRxiv

Article Title: A Long-lived Avatar for Modeling Age-Related Vascular Disease

doi: 10.64898/2026.04.29.721776

Figure Lengend Snippet: A , Representative images of iPSC-ECs in low serum (3%) and treated with VEGF, SB, or VEGF+SB (VSL) at the indicated time points reveals that VSL further improves EC morphology. The above treatments were performed using EC medium (Lonza, EGM-2MV) with 3% FBS. Ctrl* = EC medium with 3% FBS; VEGF = VEGF (10 ng/mL) added to EC medium with 3% FBS; SB = SB (SB 431542, 10 µM) added to EC medium with 3% FBS; VEGF + SB = VEGF (10 ng/mL) and SB (SB 431542, 10 µM) added to EC medium with 3% FBS, referred to as VSL. (scale bar: 100 µm) B , Quantification of cell circularity from ( A ) reveals that VSL reduces cell circularity. C , Quantification of cell area from ( A ) reveals that VSL reduces cell area. D , UMAP analysis of iPSC-ECs treated with different viability factors or combinatorial treatments of viability factors at day 40 shows that VSL restores the transcriptional profile of HAECs at day 40 to that at day 0 (n=3). The prophylactic antibiotic (Lonza Walkersville MycoZap, 0.2%; AB) was used to prevent mycoplasma contamination. D0_Ctrl = iPSC-ECs at day 0; D40_Ctrl = iPSC-ECs treated with 3%FBS EC medium (Lonza, EGM-2MV) for 40 days; D40_VEGF = iPSC-ECs treated with the 3%FBS EC medium with the addition of VEGF (10 ng/mL) for 40 days; D40_AB = iPSC-ECs treated with 3%FBS EC medium + prophylactic antibiotic (Lonza Walkersville MycoZap, 0.2%; AB) for 40 days; D40_VSL = iPSC-ECs treated with VSL for 40 days; D40_VSL+cAMP = iPSC-ECs treated with VSL and cAMP (10 µM) for 40 days; D40_VSL+cGMP = iPSC-ECs treated with VSL and cGMP (10 µM) for 40 days; D40_VSL+Camp + cGMP = iPSC-ECs treated with VSL, cAMP (10 µM), and cGMP (10 µM) for 40 days; D40_VSL + cAMP + cGMP + AB = iPSC-ECs treated with VSL, cAMP (10 µM), cGMP (10 µM) and AB (Lonza Walkersville MycoZap, 0.2%;) for 40 days. E , Representative images of β-gal staining for iPSC-ECs treated with or without VSL at day 40 are shown (scale bar: 200 µm). F , Quantification of (E) shows that VSL reduces the percentage of β-gal positive cells (n-=3). Each dot represents one single field. G , The expression of sICAM-1 and sVCAM-1 in culture media from iPSC-ECs treated with VEGF alone or VSL at day 0 or day 20 detected by ELISA assay shows that VSL effectively suppresses inflammatory cytokines (n=3). D0_Ctrl = iPSC-ECs at day 0; D20_ Ctrl = iPSC-ECs treated with standard EC medium (Lonza, EGM-2MV) for 20 days; D20_VEGF = iPSC-ECs treated with standard EC medium and addition of VEGF (10 ng/mL) for 20 days; D20_VSL = iPSC-ECs treated with VSL for 20 days. Each dot represents one technical repeat. H , Heatmap analysis of EC marker genes and fibroblast marker genes expressed in iPSC-ECs treated with or without VSL at day 60 reveals that VSL preserves EC identity during long-term culture. Each group includes triplicate in this analysis. Ctrl_D60 = iPSC-ECs treated with 3%FBS EC medium (Lonza, EGM-2MV) for 60 days. VSL_D60 = iPSC-ECs treated with VSL. Data between two groups were analyzed by Student’s t-test. Data between multiple groups are analyzed by one-way ANOVA. Results are considered statistically significant with P<0.05(*), P<0.01(**), P<0.001(***), and P<0.0001(****).

Article Snippet: HAECs were purchased from ATCC (Cat# PCS-100-011). iPSC-ECs were generated using established methods in our laboratory .

Techniques: Staining, Expressing, Enzyme-linked Immunosorbent Assay, Marker

Journal: bioRxiv

Article Title: A Long-lived Avatar for Modeling Age-Related Vascular Disease

doi: 10.64898/2026.04.29.721776

Figure Lengend Snippet: A , mVSL+ maintains co-culture of human iPSC-ECs and iPSC-VSMCs for over 180 days, with endothelial cells aligning with the lumen direction throughout the 180-day culture (scale bar: 40 µm). B , Quantification of ( A ) shows that mVSL+ preserves lumen surface integrity throughout 180-day culture. C, Immunofluorescence staining of VE-Cadherin and α-SMA in the avatars at the indicated time points (scale bar: 40 µm). D , mVSL+ maintains the expression of sICAM-1 and sVCAM-1 in the vascular avatars at a relatively low level compared to day 5 throughout the 180-day culture. (n=3). E , Bio-plex analysis shows that mVSL+ maintains the vascular avatars at a low inflammatory state compared to day 5 throughout the 180-day culture . F , The duration of prior 3D models of vascular lumens using monoculture (in red) or co-culture (in blue). Each dot represents the duration noted in one paper. 17 papers are cited in total. The large red dot indicates our monoculture duration, and the large blue dot indicates our co-culture duration.

Article Snippet: HAECs were purchased from ATCC (Cat# PCS-100-011). iPSC-ECs were generated using established methods in our laboratory .

Techniques: Co-Culture Assay, Immunofluorescence, Staining, Expressing

Journal: bioRxiv

Article Title: Molecular and genetic heterogeneity in iPSCs derived from an outbred laboratory mouse population

doi: 10.64898/2026.05.02.722403

Figure Lengend Snippet: Tail tip fibroblasts from non-sibling Diversity Outbred mice were cultured on gelatin-coated plates and reprogrammed by lentiviral transduction of a doxycycline-inducible mouse OKSM cassette. Twenty-four hours after transduction, virus-containing medium was replaced with mESM + 2i/LIF supplemented with doxycycline to induce expression of the reprogramming factors. Emerging iPSC colonies were pooled and expanded as polyclonal lines. After three passages, lines were cryopreserved at P3. One vial per line was subsequently expanded to P5 for quality control screening and genotyping. Finalized lines were banked with associated metadata in The Jackson Laboratory Biobank (see Supplemental Table 1).

Article Snippet: Supplemental Table 1 delineates the iPSC lines with associated metadata (including source fibroblast line IDs and source animal IDs) and bankstock information that can be used to obtain lines from The Jackson Laboratory.

Techniques: Cell Culture, Transduction, Virus, Expressing, Control

Journal: bioRxiv

Article Title: Molecular and genetic heterogeneity in iPSCs derived from an outbred laboratory mouse population

doi: 10.64898/2026.05.02.722403

Figure Lengend Snippet: (A) Founder haplotype frequencies across chromosomes were estimated from GigaMUGA genotypes. Contributions from each of the eight DO founder strains are shown for chromosomes 1–19 and X. Deviations from the expected 0.125 founder contribution are summarized in Supplemental Table 2. (B) Pairwise kinship coefficients were calculated from GigaMUGA-derived genotypes to assess relatedness among iPSC lines. Eight pairs of lines showed high kinship (kin_raw > 0.4; highlighted in green and arrows), and one member of each pair was flagged in the final panel . All remaining line pairs showed kinship values consistent with non-sibling Diversity Outbred mice.

Article Snippet: Supplemental Table 1 delineates the iPSC lines with associated metadata (including source fibroblast line IDs and source animal IDs) and bankstock information that can be used to obtain lines from The Jackson Laboratory.

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: Molecular and genetic heterogeneity in iPSCs derived from an outbred laboratory mouse population

doi: 10.64898/2026.05.02.722403

Figure Lengend Snippet: (A) Example of growth profiling of male and female DO iPSC lines. Cells were cultured for 96 hours and cell counts were used to calculate doubling time. Lines were assayed across multiple experimental batches. Representative colony morphologies are shown, including flattened morphology observed in some lines (e.g., line 5387, XX, 72 hours). (B) Embryoid body (EB) formation from 12 randomly selected iPSC lines. All lines formed EBs, with variation in EB morphology, some smooth and spherical and some rough and irregular, depending on the line. (C) Gene expression analysis before and after EB differentiation. RNA was collected from undifferentiated iPSCs and from EBs. Expression of pluripotency markers and early lineage markers representing ectoderm, mesoderm, and endoderm was quantified. Undifferentiated lines showed high pluripotency marker expression and low lineage marker expression, whereas EBs showed reduced pluripotency marker expression and increased lineage marker expression.

Article Snippet: Supplemental Table 1 delineates the iPSC lines with associated metadata (including source fibroblast line IDs and source animal IDs) and bankstock information that can be used to obtain lines from The Jackson Laboratory.

Techniques: Cell Culture, Gene Expression, Expressing, Marker

Journal: bioRxiv

Article Title: Molecular and genetic heterogeneity in iPSCs derived from an outbred laboratory mouse population

doi: 10.64898/2026.05.02.722403

Figure Lengend Snippet: (A) Gene expression of core pluripotency genes and early lineage markers in 218 DO iPSC lines. (B) Variance component analysis showing contributing factors to the variance in gene expression. Genotype is represented by the top 10 principal components informed by genotype probabilities.

Article Snippet: Supplemental Table 1 delineates the iPSC lines with associated metadata (including source fibroblast line IDs and source animal IDs) and bankstock information that can be used to obtain lines from The Jackson Laboratory.

Techniques: Gene Expression

Journal: bioRxiv

Article Title: Molecular and genetic heterogeneity in iPSCs derived from an outbred laboratory mouse population

doi: 10.64898/2026.05.02.722403

Figure Lengend Snippet: Quantile–quantile (Q–Q) plots of per-sample chromosome expression scores (median gene-level z-scores) were generated for each chromosome using the data from 218 DO iPSC lines. Observed values were statistically compared to the expected normal distribution. Deviation from the diagonal reflects broad chromosome-scale expression shifts. (A, B) Two examples are shown, one for a chromosome without gains or losses (Chromosome 2, A) and one for which there were frequent copy number gains (Chromosome 11, B). (C) To provide an overview for all chromosomes, the chromosome Z score was plotted for each iPSC line. Potential copy number gains were observed for Chromosomes 1, 6, 8, 11, 12, 13, and 14. Potential copy number losses were found for Chromosomes 7, 12, and 19.

Article Snippet: Supplemental Table 1 delineates the iPSC lines with associated metadata (including source fibroblast line IDs and source animal IDs) and bankstock information that can be used to obtain lines from The Jackson Laboratory.

Techniques: Expressing, Generated

Journal: bioRxiv

Article Title: Molecular and genetic heterogeneity in iPSCs derived from an outbred laboratory mouse population

doi: 10.64898/2026.05.02.722403

Figure Lengend Snippet: Tail tip fibroblasts from non-sibling Diversity Outbred mice were cultured on gelatin-coated plates and reprogrammed by lentiviral transduction of a doxycycline-inducible mouse OKSM cassette. Twenty-four hours after transduction, virus-containing medium was replaced with mESM + 2i/LIF supplemented with doxycycline to induce expression of the reprogramming factors. Emerging iPSC colonies were pooled and expanded as polyclonal lines. After three passages, lines were cryopreserved at P3. One vial per line was subsequently expanded to P5 for quality control screening and genotyping. Finalized lines were banked with associated metadata in The Jackson Laboratory Biobank (see Supplemental Table 1).

Article Snippet: The remaining 296 polyclonal iPSC lines were frozen at P3, genotyped, and expanded to P5-P6 for QC and biobanking at The Jackson Laboratory ( , Supplemental Table 1).

Techniques: Cell Culture, Transduction, Virus, Expressing, Control

Journal: bioRxiv

Article Title: Molecular and genetic heterogeneity in iPSCs derived from an outbred laboratory mouse population

doi: 10.64898/2026.05.02.722403

Figure Lengend Snippet: (A) Founder haplotype frequencies across chromosomes were estimated from GigaMUGA genotypes. Contributions from each of the eight DO founder strains are shown for chromosomes 1–19 and X. Deviations from the expected 0.125 founder contribution are summarized in Supplemental Table 2. (B) Pairwise kinship coefficients were calculated from GigaMUGA-derived genotypes to assess relatedness among iPSC lines. Eight pairs of lines showed high kinship (kin_raw > 0.4; highlighted in green and arrows), and one member of each pair was flagged in the final panel . All remaining line pairs showed kinship values consistent with non-sibling Diversity Outbred mice.

Article Snippet: The remaining 296 polyclonal iPSC lines were frozen at P3, genotyped, and expanded to P5-P6 for QC and biobanking at The Jackson Laboratory ( , Supplemental Table 1).

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: Molecular and genetic heterogeneity in iPSCs derived from an outbred laboratory mouse population

doi: 10.64898/2026.05.02.722403

Figure Lengend Snippet: (A) Histogram of kinship values estimated from genotype probabilities across the full DO iPSC panel. Each bar represents the frequency of pairwise comparisons between distinct lines. Most comparisons are tightly centered around K ≈ 0.15 (median = 0.151; mean = 0.154), consistent with the expected relatedness structure of a multiparent Diversity Outbred population. Ninety-five percent of pairs fall below K = 0.171 and 99% below K = 0.230. A small number of pairs are in the extreme upper tail (K > 0.50), approaching the diagonal kinship value expected for genetically identical samples. (B, C) Minor allele frequency (MAF) and heterozygosity (H) were evaluated in the DO iPSC panel and across all measures the population structure of this panel is comparable to a similarly sized genetic mapping cohort of non-sibling DO mice with median MAF = 0.278, mean expected heterozygosity = 0.367 and mean observed = 0.364

Article Snippet: The remaining 296 polyclonal iPSC lines were frozen at P3, genotyped, and expanded to P5-P6 for QC and biobanking at The Jackson Laboratory ( , Supplemental Table 1).

Techniques:

Journal: bioRxiv

Article Title: Molecular and genetic heterogeneity in iPSCs derived from an outbred laboratory mouse population

doi: 10.64898/2026.05.02.722403

Figure Lengend Snippet: (A) Example of growth profiling of male and female DO iPSC lines. Cells were cultured for 96 hours and cell counts were used to calculate doubling time. Lines were assayed across multiple experimental batches. Representative colony morphologies are shown, including flattened morphology observed in some lines (e.g., line 5387, XX, 72 hours). (B) Embryoid body (EB) formation from 12 randomly selected iPSC lines. All lines formed EBs, with variation in EB morphology, some smooth and spherical and some rough and irregular, depending on the line. (C) Gene expression analysis before and after EB differentiation. RNA was collected from undifferentiated iPSCs and from EBs. Expression of pluripotency markers and early lineage markers representing ectoderm, mesoderm, and endoderm was quantified. Undifferentiated lines showed high pluripotency marker expression and low lineage marker expression, whereas EBs showed reduced pluripotency marker expression and increased lineage marker expression.

Article Snippet: The remaining 296 polyclonal iPSC lines were frozen at P3, genotyped, and expanded to P5-P6 for QC and biobanking at The Jackson Laboratory ( , Supplemental Table 1).

Techniques: Cell Culture, Gene Expression, Expressing, Marker

Journal: bioRxiv

Article Title: Molecular and genetic heterogeneity in iPSCs derived from an outbred laboratory mouse population

doi: 10.64898/2026.05.02.722403

Figure Lengend Snippet: (A) Gene expression of core pluripotency genes and early lineage markers in 218 DO iPSC lines. (B) Variance component analysis showing contributing factors to the variance in gene expression. Genotype is represented by the top 10 principal components informed by genotype probabilities.

Article Snippet: The remaining 296 polyclonal iPSC lines were frozen at P3, genotyped, and expanded to P5-P6 for QC and biobanking at The Jackson Laboratory ( , Supplemental Table 1).

Techniques: Gene Expression

Journal: bioRxiv

Article Title: Molecular and genetic heterogeneity in iPSCs derived from an outbred laboratory mouse population

doi: 10.64898/2026.05.02.722403

Figure Lengend Snippet: Quantile–quantile (Q–Q) plots of per-sample chromosome expression scores (median gene-level z-scores) were generated for each chromosome using the data from 218 DO iPSC lines. Observed values were statistically compared to the expected normal distribution. Deviation from the diagonal reflects broad chromosome-scale expression shifts. (A, B) Two examples are shown, one for a chromosome without gains or losses (Chromosome 2, A) and one for which there were frequent copy number gains (Chromosome 11, B). (C) To provide an overview for all chromosomes, the chromosome Z score was plotted for each iPSC line. Potential copy number gains were observed for Chromosomes 1, 6, 8, 11, 12, 13, and 14. Potential copy number losses were found for Chromosomes 7, 12, and 19.

Article Snippet: The remaining 296 polyclonal iPSC lines were frozen at P3, genotyped, and expanded to P5-P6 for QC and biobanking at The Jackson Laboratory ( , Supplemental Table 1).

Techniques: Expressing, Generated

Journal: bioRxiv

Article Title: Molecular and genetic heterogeneity in iPSCs derived from an outbred laboratory mouse population

doi: 10.64898/2026.05.02.722403

Figure Lengend Snippet: (A) Genomic locations of DO iPSC eQTLs (LOD > 7.78). Red arrow denotes chromosome 15 eQTL hotspot with over 600 distant target genes. (B) Distant eQTL hotspots, where distant is defined as eQTL greater than 2Mbp from its target gene. Asterisks denote hotspots shared with published DO ESC transcriptomics. (C) Heat map of mediation analysis identifying Lifr as the strongest mediator for the chromosome 15 hotspot. Potential mediators were identified using the change in LOD score and the associated adjusted pvalues for each mediator-target interaction are represented by color. (D) Haplotype effect at the local eQTL for Lifr showing a 3:5 split, with more recently wild-derived strains grouping separately from the classic inbred strains. (E) Heatmap of the haplotype effects at the 696 distant eQTL within the chromosome 15 hotspot showing a 3:5 split between the wild-derived and classic inbred strains.

Article Snippet: The remaining 296 polyclonal iPSC lines were frozen at P3, genotyped, and expanded to P5-P6 for QC and biobanking at The Jackson Laboratory ( , Supplemental Table 1).

Techniques: Transcriptomics, Derivative Assay