Comparative Analysis of Vascular Cell Differentiation From Peripheral Blood Mononuclear Cell- and Urine-Derived Induced Pluripotent Stem Cells

Abstract

Background and Objectives: Peripheral blood mononuclear cells (PBMCs) and urine-derived epithelial cells have both emerged as valuable sources for induced pluripotent stem cell (iPSC) generation, each presenting unique advantages in terms of accessibility and reprograming efficiency. This study aimed to assess and compare the potential of PBMC-derived iPSCs (PiPSCs) and urine-derived iPSCs (UiPSCs) in generating functional endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), which are critical for vascular tissue engineering and disease modeling. Phenotypic characteristics, differentiation efficacy, and functional properties of iPSC-derived ECs and VSMCs from these distinct sources were investigated to reveal variations attributed to cellular origin.

Methods and Results: PiPSCs and UiPSCs both successfully differentiated into functional ECs and VSMCs. EC differentiation efficiency was similar, yielding about 45% mature ECs with characteristic morphology, marker expression, and tube formation abilities, showing no significant differences between cell types. Transcriptomic analysis revealed upregulation of key endothelial markers (platelet endothelial cell adhesion molecule 1 [PECAM1], cadherin 5 [CDH5], and melanoma cell adhesion molecule [MCAM]) and downregulation of lymphatic markers (Fms-related tyrosine kinase 4 [FLT4], prospero homeobox protein 1 [PROX1], and podoplanin [PDPN]), confirming blood EC identity. The upregulation of collagen type IV alpha 1 chain (COL4A1) and collagen type I alpha 1 chain (COL1A1) indicated a mature endothelial state with enhanced extracellular matrix (ECM) production. VSMC differentiation resulted in high percentages of α-smooth muscle actin (α-SMA) positive cells for both PiPSCs (96%) and UiPSCs (94%). These VSMCs exhibited typical spindle-shaped morphology, expressed VSMC markers, and responded to carbachol. Transcriptomic analysis showed significant upregulation of VSMC markers (actin alpha 2 [ACTA2], caldesmon 1 [CALD1], calponin 1 [CNN1], transgelin [TAGLN], tropomyosin 2 [TPM2]), with concurrent downregulation of ACTA1 and upregulation of ACTA2, confirming their vascular smooth muscle phenotype. The upregulation of COL6A1 in VSMCs indicated a mature phenotype with enhanced ECM production, crucial for vascular tissue integrity and function. Gene set enrichment analysis highlighted the upregulation of multiple hallmark pathways, delineating a distinctive transcriptional profile.

Conclusions: This study presents a comprehensive comparative analysis of functionally differentiated ECs and VSMCs derived from PiPSCs and UiPSCs, providing critical insights into the expression patterns and phenotypic transitions during differentiation. Our findings enhance the understanding of distinct molecular signatures in iPSCs from different sources and their progeny.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

Induced pluripotent stem cells (iPSCs) are generated from mature, fully differentiated somatic cells that have been reprogramed through the introduction of a key set of genes, thereby generating a pluripotent cell line [1, 2]. These cells are defined by their infinite proliferation capacity and the ability to differentiate into specialized cell types of all three germ layers [3]. IPSCs display properties of embryonic stem cells (ESCs) regarding morphology, proliferation, surface antigens, gene expression, and epigenetic status of pluripotent cell-specific genes. Their discovery has resolved major obstacles accompanied by ESCs circumventing the ethical concerns involved in the use of ESCs as the derivation involves somatic cells instead of early human blastocysts [1].

The first report on the generation of iPSCs using mouse fibroblasts was published in 2006 by Takahashi and Yamanaka [2]. Since then, the technique has successfully been applied to various other cell types, including human dermal fibroblasts [3], adipose stem cells [4], neural progenitor cells [5, 6], hepatocytes and gastric epithelial cells [7], peripheral blood mononuclear cells (PBMCs) [8, 9], and urine-derived epithelial cells [10]. PBMCs and urine-derived epithelial cells both offer promising sources for clinical and research applications given their ease of harvest. Due to minimally invasive and noninvasive procedures, they can be repeatedly isolated from patients regardless of their diagnosis. This provides a significant advantage over other cell sources, which may require invasive procedures for repeated collection [10, 11].

Reprograming of somatic cells into a pluripotent state allows the generation of patient- and disease-specific stem cells, harboring a great application potential in human cell-based therapy, regenerative medicine [12, 13], disease modeling [14], and drug screening [15]. It has been demonstrated that iPSCs are capable of differentiation into vascular cell phenotypes in response to defined media and culture conditions providing an autologous and abundant source for application in these fields [16]. IPSC-derived endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) harbor a potential for studying cardiovascular diseases, such as the use of patient-cell–based tissue engineered blood vessels. In particular, the advances in microfluidics technologies and personalized medicine may serve as a basis for the implementation of the iPSC technology in broad-scale personalized vascular disease modeling approaches. ECs develop from the mesoderm and cover the luminal surface of all vessels. Key functions of the endothelial barrier in blood vessels include preventing clotting activation of the coagulation cascade and leukocyte adhesion, protecting vascular interstitial cells from direct shear stress, and regulating the transport of oxygen, nutrients, and fluids between blood and tissues [17, 18]. VSMCs represent the major cell population of the tunica media of arteries and arterioles. They are crucial for structural support, production, and remodeling of the extracellular matrix (ECM), as well as contraction and relaxation of the vessel in response to EC-driven nitrogen oxide signaling [19–21]. A range of protocols for differentiating murine and human iPSCs into functional ECs [22, 23] and vascular VSMCs [24, 25] have been published, with ongoing efforts to further delineate more efficient methods to obtain an increasing yield of proliferative mature vascular cells. However, especially iPSC-derived ECs exhibit a broad functional heterogeneity [26].

This study aimed to assess and compare the potential of PBMC-derived iPSCs (PiPSCs) and urine-derived iPSCs (UiPSCs) in generating functional ECs and VSMCs. Examining and comparing the phenotypic characteristics, differentiation efficacy, and functional properties of iPSC-derived ECs and VSMCs from these distinct sources can reveal variations attributed to cellular origin, potentially highlighting critical differences that warrant customized differentiation protocols. By investigating these aspects, this study aims to uncover insights into optimizing iPSC-derived vascular cells for therapeutic applications, particularly in cardiovascular diseases and personalized medicine strategies.

2. Materials and Methods

A supplementary materials and methods section is provided in Supporting Information.

2.1. Reprograming of PBMCs to iPSCs

PBMCs were obtained from one healthy male. Following the isolation of PBMCs from whole blood, erythroblasts were expanded using StemSpan Medium (Stemcell Technologies, Canada) containing 50 U/mL penicillin/streptomycin (Gibco, United States), 50 µg/mL ascorbic acid, 3 U/mL erythropoietin, 100 ng/mL stem cell factor, 10 ng/mL interleukin 3, 1 µM dexamethasone, and 40 ng/mL insulin growth factor 1. Cells were incubated under standard conditions (37°C, 95% humidity, 5% CO₂) for 9 days. Medium changes were performed every 3 days. After 9 days of expansion, flow cytometry analysis was performed to ensure a CD71 (BD Biosciences, United States) positive, CD14 (BD Biosciences, United States) negative cell population.

Transduction of erythroblasts was performed using the Sendai virus reprograming kit CytoTune iPS 2.0 (Invitrogen, United States) according to the manufacturer’s instructions. On day 3 after transduction, cells were switched to six-well feeder plates coated with 1% Matrigel hESC-qualified Matrix (Corning, United States). On day 4, media was changed to advanced Dulbecco’s Modified Eagle Medium (DMEM)/F12 (Gibco, United States) supplemented with 20% Knock out serum, 1 mM L-glutamine, 50 U/mL penicillin/streptomycin, 0.1% 2-mercaptoethanol (all Gibco, United States), and 4 µg/mL fibroblast growth factor (Merck, Germany) and was changed every day until colonies were ready to be picked. Colony picking was performed 20 days after transduction and cells were transferred to Matrigel-coated 96-well plates and treated with mTeSR plus medium including supplements (Stemcell Technologies, Canada). UiPSCs were kindly provided by Evercyte (Vienna, Austria). Cells were routinely tested for the absence of bacterial (including mycoplasma), fungal, and viral (human immunodeficiency virus [HIV], hepatitis A virus [HAV], hepatitis B virus [HBV], hepatitis C virus [HCV], Parvovirus B19) contaminations.

After colony expansion of UiPSCs and PiPSCs, flow cytometry analysis for stage-specific embryonic antigen-4 (SSEA-4, BD Biosciences, United States) was performed to ensure pluripotency. Additionally, the expression of the pluripotency markers octamer-binding transcription factor (OCT4), sex-determining region Y-box 2 (SOX2), Kruppel-like factor 4 (KLF4), and c-Myc was assessed using immunocytochemistry, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and western blot.

Methods for flow cytometry, immunofluorescence, and qRT-PCR analyses are found in detail in the Materials and Methods section in the Supporting Information.

2.2. Differentiation of iPSCs to ECs

For EC differentiation, embryoid body formation was performed for 4–5 days prior to treatment with small molecules and EC growth medium. For the formation of embryoid bodies, cells were transferred to a six-well culture dish without coating adding mTeSR Plus medium supplemented with 10 µM Rock Inhibitor Y-27632 (Enzo Life Sciences, United States). The first medium change was performed after 48 h to allow for embryoid body formation, after confirming appropriate morphology through light microscopy. Afterward, medium changes were performed daily until embryoid bodies reached a diameter of 150–200 µm, which occurred after 4–5 days.

Embryoid bodies were then transferred to a 10 cm culture dish containing Roswell Park Memorial Institute (RPMI) medium containing B-27 supplement (both Gibco, United States). Small-molecule inhibitor of glycogen synthase kinase-3β (CHIR99021, Sigma–Aldrich, United States) was added at a concentration of 12 µM. Additionally, 10 µM rock inhibitor was added to prevent apoptosis. After 24 h, CHIR99021 was removed and cells were treated with RPMI/B27 medium without further supplements for another 24 h. On day 3, RPMI/B-27 medium was changed and supplemented with 10 ng/mL BMP4 (Fischer Scientific, United States), 10 µM SB43 (Sigma–Aldrich, United States), 10 µM purmorphamine (Sigma–Aldrich, United States) and 100 ng/mL VEGF-A (Peprotech, United States). On day 5, the culture medium was switched to EC growth medium-2 (EGM-2, Promocell, Germany) supplemented with 10 ng/mL BMP4 (Merck, Germany) and 100 ng/mL VEGF-A (Merck, Germany) for 48 h. On day 7, cells were transferred to 1% gelatin-coated culture dishes and treated with EGM-2 supplemented with 100 ng/mL VEGF-A for another 48 h. To increase the purity of derived ECs, magnetic-activated cell sorting (MACS) for the cell-type–specific surface marker CD31 was performed on day 9. Flow cytometry analysis for CD31 on iPSC-derived ECs was performed before and after MACS. Additionally, to ensure the loss of pluripotency markers, flow cytometry analysis for stage-specific embryonic antigen-4 (SSEA-4) was performed before and after differentiation. Human umbilical vein endothelial cells (HUVECs) served as control.

For characterization, we performed immunofluorescence and RT-qPCR for the EC markers CD31, van Willebrand factor (VWF), and VE-cadherin. HUVECs served as control.

2.3. Tube Formation Assay

To assess and compare the functional properties of iPSCs-derived ECs, tube formation assays were performed. Prior to the experiment, cells were temporarily labeled with the transient fluorescent dye CellTracker Green (Thermo Fisher Scientific, United States). An eight-well µ-slide (ibidi, Planegg, Germany) was coated with 80 µL Matrigel per well and placed in an incubator with standard culture conditions for 20 min for polymerization. Subsequently, a total of 4 × 10⁴ ECs were transferred to each well. Tube formation was assessed after 8 and 24 h using a confocal microscope (Zeiss, Germany). Vessel sprouting, total tube length, and the number of branch points were assessed. HUVECs served as control.

2.4. Differentiation of iPSCs to VSMCs

VSMC differentiation was performed with a monolayer culture. Cells were treated with RPMI media (Gibco, United States) supplemented with B27 (Gibco, United States), 12 µM small-molecule CHIR99021 (Sigma–Aldrich, United States), and 10 ng/mL BMP4 (Merck, Germany) for 3 days followed by 1 day of RPMI/B27 without further supplements. Afterward, cells were treated with a differentiation medium based on high glucose DMEM (Gibco, United States) supplemented with 10% fetal calf serum (FCS), 5% horse serum (Gibco, United States), 100 mM nonessential amino acids (Gibco, United States), 100 mM 2-mercaptoethanol (Gibco, United States), 1 mM L-glutamine (Gibco, United States), and 50 U/mL penicillin/streptomycin (Gibco, United States) for 6 days, followed by VSMC growth medium-2 including supplements (Lonza, Switzerland) for 6 days. Medium was then again changed to high glucose DMEM supplemented with 10% FCS, 1 mM L-glutamine, and 50 U/mL penicillin/streptomycin for another 6 days. Flow cytometry analysis for α-smooth muscle actin (α-SMA) was performed to confirm differentiation to VSMCs. To ensure the loss of pluripotency markers, flow cytometry analysis for SSEA-4 was performed before and after differentiation.

For characterization, immunofluorescence and RT-qPCR for the VSMC markers α-SMA, calponin 1, and transgelin were performed. Primary VSMC served as control. Additionally, immunofluorescence and RT-qPCR were performed for the pluripotency markers SOX2, KLF4, OCT4, and c-Myc. UiPSC and PiPSC served as control.

2.5. Contractility Assay

To evaluate the functional properties of differentiated VSMCs, the contractility was assessed using carbachol. Cells were stained with the transient fluorescent dye Cell Tracker Green (Thermo Fisher Scientific, United States) 24 h prior to the assay and seeded on a six-well plate coated with 1% gelatin. Subsequently, cells were treated with nonenzymatic releasable surface reagent (ReLeSR) (Stemcell Technologies, Canada) for 30 s followed by incubation under standard cell culture conditions for 2 min and 30 s to achieve partial detachment. Once the cells were partially detached, cold DMEM high glucose medium containing Carbamoylcholinchlorid (Merck, Germany) at a final concentration of 1 µM was added. After 5 min of incubation, the solution was carefully removed and fresh DMEM high glucose medium was added to the cells. Contraction of the cells was analyzed by comparing the cell surface area before and 0, 2, 4, and 5 min after treatment. Images were obtained with a confocal microscope (LSM980 Zeiss, Germany). Primary VSMCs served as positive and HUVECs as negative control.

2.6. Statistical Analysis

Data are presented as mean ± standard error (SE) unless otherwise specified. Statistical testing was conducted where appropriate to assess the significance of observed differences. To compare the means of multiple groups and determine if they were significantly different, one-way analysis of variance (ANOVA) was employed. Post hoc tests, such as Tukey’s honestly significant difference (HSD) test, were applied for pairwise comparisons when the ANOVA indicated significant differences among groups. Statistical significance was set at a predetermined alpha level of 0.05. All analyses were performed using GraphPad Prism Version 9.3.1.

2.7. RNA Sequencing and Data Analysis

Sequencing libraries from total RNA were prepared at the Core Facility Genomics, Medical University of Vienna, using the QuantSeq 3′ forward (FWD) protocol Version 1 (Lexogen). Eighteen PCR cycles were used for library prep, as determined by qPCR according to the library prep manual. Libraries were quality control (QC)-checked on a Bioanalyzer 2100 (Agilent) using a High Sensitivity DNA Kit for correct insert size and quantified using Qubit dsDNA HS Assay (Invitrogen). Pooled libraries were sequenced on a P1 flowcell on a NextSeq2000 instrument (Illumina) in 1x75bp single-end sequencing mode.

RNA sequencing was performed with four biological replicates of each cell type. On average, 5 million reads per sample were generated. Reads in fastq format were generated using the Illumina bcl2fastq command line tool (v2.19.1.403). Reads were trimmed and filtered using cutadapt [27] Version 2.8 to trim polyA tails, remove reads with N′s and trim bases with a quality of less than 30 from the 3′ ends of the reads. On average, 4 million reads were left after this procedure.

Trimmed reads in fastq format were aligned to the human reference genome version GRCh38 [28] with Gencode 29 annotations [28] using STAR aligner [29] Version 2.6.1a in 2-pass mode. Raw reads per gene were counted by STAR. Differential gene expression was calculated using DESeq2 Version 1.22.2 [30].

Volcano plots, generated using VolcaNoseR [31], provide a visually informative representation of the differential gene expression landscape. Heat maps were constructed using R [32] and ComplexHeatmap v2.18.0 [33] highlighting the 25 most up- and downregulated pathways specific to each cell type as Z-scores. Additionally, heat maps were generated to compare the expression of pluripotency markers and markers specific to VSMCs or ECs between iPSCs and their derived VSMCs or ECs. Gene set enrichment analysis (GSEA) was executed using GSEA software Version 4.3.2, to assess the enrichment or depletion of defined gene sets. Each gene set is generated from numerous “founder” sets and represents a particular biological state or process. By minimizing both variation and redundancy, these hallmarks efficiently condense the majority of the pertinent data and offer more concise inputs for gene set enrichment research [34–36]. Transcriptomic analysis results were further validated with RT-qPCR analysis.

3. Results

3.1. Reprograming of PBMCs to iPSCs

PBMCs were successfully reprogramed to iPSCs. Prior to reprograming, flow cytometry was performed to ensure a cell population that is CD71 positive, which is strongly expressed in erythroid precursor cells and CD14, which is mainly expressed in macrophages and monocytes, was negative (Figure S1).

PiPSC and UiPSC both exhibited dome-shaped stem cellular morphology and expressed the pluripotency markers OCT4, SOX2, and KLF4 (Figure 1a). Flow cytometry analysis proved expression of the pluripotency marker SSEA-4 (Figure 1b). The presence of OCT4, SOX2, and KLF4 as well as the absence of the EC marker CD31 and the VSMC marker α-SMA was demonstrated by western blot analysis (Figure 1c). The expression of pluripotency markers on the mRNA level was confirmed by RT-qPCR (Figure 1d). Prior to differentiation, no differences in expression pattern or cellular morphology between PiPSCs and UiPSCs could be detected.

[figure(s) omitted; refer to PDF]

3.2. Differentiation of iPSCs to Functional ECs

PiPSCs and UiPSCs were successfully differentiated to ECs. Differentiation was achieved by embryoid body formation followed by in vitro monolayer culture treated with defined culture media supplemented with factors guiding toward the development of an EC phenotype (Figure 2a). This process yielded a cell population containing 44.1% (±0.7) CD31 positive cells in case of PiPSCs and 46% (±3.0) for UiPSCs ($p=0.36$, Figure 2b). Following the differentiation process, cells were sorted once by MACS for CD31 positivity, which increased the fraction of CD31-positive cells to 96.1% (±0.9) for UiPSC-derived ECs and 96.4% (±0.7) for PiPSC-derived ECs (Figure 2c). Derived ECs of both origins did no longer express the pluripotency marker SSEA-4 (Figure 2c). PiPSC-derived ECs and UiPSC-derived ECs both exhibited a cobblestone-like pattern and expressed the EC markers CD31, VE-cadherin (CD144), and vWF, which could be shown by immunofluorescence staining (Figure 2d). The expression of CD31, VE-cadherin, and vWF was also assessed on RNA level by RT-qPCR analysis, which showed upregulation of all three EC markers compared to HUVECs, indicating an activated EC phenotype. In general, the expression of pluripotency genes (OCT4, KLF4, and c-Myc) was downregulated. However, PiPSC ECs and UiPSC ECs continued to show an increased expression of SOX2 compared to HUVECs (Figure 2e).

[figure(s) omitted; refer to PDF]

3.3. Tube Formation Assay Demonstrates Functionality of Derived ECs

Tube formation assays proved the functionality of iPSC-derived ECs demonstrating the formation of tube-like structures connected at branch points of PiPSC-derived ECs and UiPSC-derived ECs (Figure 3a). PiPSC-derived ECs and UiPSC-derived ECs showed no significant difference in total tube length ($p=0.86$) and number of branch points ($p=0.20$) after 8 h. However, after 24 h UiPSC-derived ECs had built a significantly higher number of tubes ($p=0.04$). The number of branch points did not differ significantly ($p=0.55$). HUVECs, which served as a positive control, had built a significantly higher number of tubes after 8 h compared to PiPSC-derived ECs ($p<0.0001$) and UiPSC-derived ECs ($p<0.0001$). After 24 h of incubation with standard culture conditions, the created tube length did not differ significantly between HUVECs and iPSC-derived ECs. The tube formation assay using HUVECs showed a significantly higher number of branch points compared to PiPSC-derived ECs ($p<0.0001$) and UiPSC-derived ECs ($p<0.0001$) after 8 h of incubation. After 24 h, HUVECs continued to have built more branch points compared to UiPSC-dervied ECs ($p=0.02$). The difference between PiPSC-derived ECs and HUVECs for this matter was not statistically significant ($p=0.39$, Figure 3b,c).

[figure(s) omitted; refer to PDF]

3.4. Differentiation of iPSCs to Functional VSMCs

PiPSCs and UiPSCs were successfully differentiated into VSMCs using a monolayer culture approach (Figure 4a). For PiPSCs, the differentiation process yielded a cell population containing 93.7% (±1.1) α-SMA positive cells. Regarding UiPSCs 95.7% (±1.7) of derived cells were α-SMA positive. For comparison, primary VSMCs served as control cells and were α-SMA positive in 99.4% (±0.4). The difference in α-SMA positive cells in the population of PiPSC-derived VSMCs and UiPSC-derived VSMCs was not statistically significant ($p=0.17$, Figure 4b,c). IPSC-derived VSMCs did not express the pluripotency marker SSEA-4 (Figure 4b). Due to the high output of α-SMA positive cells, further sorting using MACS was not necessary. IPSC-derived VSMCs showed a spindle-like structure and expressed the VSMC markers α-SMA, calponin 1, and transgelin (Figure 5a). RT-qPCR analysis demonstrated higher expression of α-SMA, calponin 1, and transgelin in iPSC-derived VSMCs compared to primary VSMCs, however, without differences between cells derived from PiPSCs and UiPSCs. The pluripotency marker KLF4 was downregulated. However, the pluripotency markers SOX2, OCT4, and c-Myc were expressed to a higher extent in VSMCs derived from both iPSC sources when compared to primary VSMCs (Figure 5b).

[figure(s) omitted; refer to PDF]

3.5. Derived VSMCs Show Adequate Contractility in Response to Carbachol

PiPSC-derived VSMCs and UiPSC-derived VSMCs both demonstrate adequate contractility in response to carbachol. The amount of contraction, measured as a change in the cell surface area, was assessed. After carbachol treatment, the cell surface area of PiPSC VSMCs, UiPSC VSMCs, and VSMCs diminished to 53.6% (±22.8), 57.5% (±23.2), and 32.3% (±2.6), respectively. The cell surface area of HUVECs remained at 100% (±13.9) (Figure 5c).

3.6. Comparative Transcriptomic Analysis of iPSCs Unravels Differential Gene Expression Patterns

Comparative transcriptomic analysis of iPSCs, reprogramed from urine-derived cells and PBMCs, unveiled differential gene expression patterns in both cell types (Figure 6a,b).

[figure(s) omitted; refer to PDF]

Subsequent GSEA delineated a distinctive transcriptional profile marked by the upregulation of multiple hallmark pathways. In PiPSCs, pathways associated with cell cycle progression, such as E2F targets and Myc targets, displayed increased expression, indicating a heightened potential for cellular growth and proliferation. Metabolic pathways, including MTORC1 signaling, oxidative phosphorylation, and glycolysis, were also significantly upregulated, suggesting an enhanced capacity for energy production and nutrient utilization. Concurrently, pathways related to oxidative stress response and cellular repair mechanisms exhibited heightened expression. Furthermore, signaling pathways associated with angiogenesis were activated (Figure 6c). In UiPSCs, pathways associated with cell–cell adhesion and tissue structure, such as apical junction and epithelial–mesenchymal transition, displayed increased expression, suggesting potential alterations in cellular morphology and adhesion dynamics. The downregulation of Kirsten rat sarcoma viral oncogene homolog (KRAS) signaling implies a dampened activity in pathways related to cell growth and survival (Figure 6d).

3.7. Comparative Transcriptomic Analysis of VSMCs Derived From PiPSCs and UiPSCs

In our exploration of the transcriptomic landscape of VSMCs derived from PiPSCs and UiPSCs, a detailed analysis revealed a range of up- and downregulated pathways, offering insights into the molecular identity of these cells (Figure 7a,b).

[figure(s) omitted; refer to PDF]

PiPSC VSMCs showed an increased engagement of KRAS-mediated signaling pathways, a key component of intracellular signaling networks involved in the regulation of cell growth, survival, and differentiation. Additionally, the upregulation of the mitotic spindle pathway suggested pronounced changes in cell division dynamics, potentially influencing the proliferative capacity of these cells. Simultaneously, the activation of myogenesis pointed toward active muscle cell differentiation processes, aligning with the phenotypic identity of smooth muscle cells (Figure 7c).

In UiPSC VSMCs, the heightened expression of Myc targets suggests an active engagement of Myc-mediated signaling pathways, implying a propensity for cellular proliferation and growth. Additionally, the upregulation of E2F targets points toward an enhanced involvement in cell cycle regulation and DNA synthesis. Concurrently, the upregulation of oxidative phosphorylation indicates an increased reliance on mitochondrial energy production, reflecting the energetic demands associated with the heightened cellular activity in these VSMCs. Furthermore, the pro-angiogenic phenotype indicated by the upregulation of angiogenesis suggests a potential role of UiPSC-derived VSMCs in supporting vascularization (Figure 7d).

As the next step, we analyzed the raw gene counts of VSMC markers and pluripotency markers, comparing iPSCs and their derived VSMCs. The findings were validated using RT-qPCR analysis of selected VSMC and pluripotency markers. The analysis demonstrated a distinct upregulation of VSMC marker expression, specifically actin alpha 2 (ACTA2), caldesmon 1 (CALD1), calponin 1 (CNN1), transgelin (TAGLN), and tropomyosin 2 (TPM2), in VSMCs derived from both PiPSCs and UiPSCs. Furthermore, ACTA1 expression was downregulated in both VSMC types, whereas ACTA2 was upregulated, confirming the vascular smooth muscle phenotype since ACTA1 is predominantly expressed in skeletal muscle and ACTA2 in vascular smooth muscle [37, 38]. Additionally, the raw gene counts revealed a notable upregulation of collagen type VI alpha 1 Chain (COL6A1) in VSMCs, indicating a shift toward a more mature phenotype capable of ECM production, which is essential for vascular tissue function and integrity [39–41]. Conversely, pluripotency markers such as SOX2 were markedly downregulated (Figure 8a). These results were corroborated by RT-qPCR, which showed that ACTA2, TAGLN, CNN1, and SOX2 are differentially regulated in PiPSC- and UiPSC-derived VSMCs compared to their iPSC origins (Figure 8b–e).

[figure(s) omitted; refer to PDF]

3.8. Comparative Transcriptomic Analysis of ECs Derived From PiPSCs and UiPSCs

Transcriptomic analysis of ECs derived from PiPSCs and UiPSCs unveiled a distinct molecular profile (Figure 9a,b).

[figure(s) omitted; refer to PDF]

In PiPSC ECs, the activation of the P53 pathway implies potential regulatory roles in cell cycle control and responses to DNA damage, contributing to genomic stability in these cells. Notably, the downregulation of KRAS signaling indicates a subdued signaling environment related to cell growth. The upregulation of angiogenesis underscores the pro-angiogenic phenotype of PiPSC ECs, suggesting their involvement in vascularization processes. Additionally, the activation of glycolysis points toward enhanced energy production through glycolytic pathways (Figure 9c).

The transcriptomic analysis of ECs derived from UiPSC revealed elevated expression of Myc targets and mitotic spindle pathways, indicative of a proliferative, and growth-oriented cellular state. Additionally, the increased reliance on oxidative phosphorylation highlights a metabolic adaptation, supporting the energetic demands associated with the active cellular processes (Figure 9d).

Following this, we analyzed the raw gene counts of EC markers and pluripotency markers, comparing the profiles of iPSCs and their derived ECs. The results were validated through RT-qPCR analysis of selected EC and pluripotency markers. The data revealed a notable upregulation of EC marker expression, for example, platelet EC adhesion molecule 1 (PECAM1), VE-cadherin (CDH5), and melanoma cell adhesion molecule (MCAM), in ECs derived from both PiPSCs and UiPSCs. Furthermore, lymphatic EC markers like Fms-related tyrosine kinase 4 (FLT4), prospero homeobox protein 1 (PROX1), and podoplanin (PDPN) were downregulated in the derived ECs, indicating that these cells are indeed blood ECs and not lymphatic ECs [42, 43]. The upregulation of collagen type IV alpha 1 chain (COL4A1) and collagen type I alpha 1 chain (COL1A1) in ECs compared to iPSCs suggests a phenotypic transition toward a more mature EC state, with enhanced ECM production [44, 45]. In contrast, pluripotency markers such as SOX2 were distinctly downregulated. (Figure 10a). These results were confirmed by RT-qPCR, which showed that CD31, VE-cadherin, and SOX2 are differentially regulated in PiPSC- and UiPSC-derived ECs compared to their iPSC origins (Figure 10b–d). Kinase insert domain receptor (KDR), also known as vascular endothelial growth factor receptor 2 (VEGFR-2), exhibited similarly low expression levels in both iPSCs and derived ECs (Figure 10a,e).

[figure(s) omitted; refer to PDF]

4. Discussion

The advancement of iPSC technologies provides a promising cell source for tissue engineering and regenerative medicine applications offering differentiation capacities into all cell types of the body with unlimited scale-up potential [19]. Since the first report on the generation of iPSCs by Takahashi and Yamanaka [2], the technology has gained increasing interest, offering the ability to create patient- and disease-specific stem cells for a wide variety of applications in regenerative medicine [12, 13], disease modeling [14], and drug screening [15]. In this respect, PBMCs and urine-derived epithelial cells have both emerged as valuable sources for iPSC generation, each presenting unique advantages in terms of accessibility and reprograming efficiency [8–11].

We have successfully reprogramed PBMCs to iPSCs using a Sendai virus vector, a nonsegmented negative-strand RNA virus that does not integrate into the cellular DNA and induces cytoplasmic gene expression. The advantage of this virus vector system lies in the capability to express the reprograming genes without chromosomal integration, thereby circumventing the associated risks including gene disruption, oncogenesis, and premature cell death as a result of insertional mutagenesis [46, 47].

We have demonstrated successful differentiation of PiPSCs and UiPSCs to functional ECs and VSMCs. We did not observe differences between the iPSC origins, regarding differentiation, morphology, or functionality of both vascular cell types. The EC phenotype was induced via embryoid body formation thereby bypassing an intermediate mesodermal state mimicking early embryonic development [48]. The efficacy of the differentiation process did not differ between PiPSCs and UiPSCs yielding approximately 45% mature ECs in both cases. Derived ECs displayed morphological and functional characteristics consistent with primary ECs. Functionality of derived ECs was demonstrated via tube formation assays, showing no significant difference regarding the amount of newly formed tubes and branches between PBMC-derived ECs and UiPSC-derived ECs. Transcriptomic analysis provided deeper insight into the molecular characteristics, demonstrating the upregulation of key endothelial markers, including PECAM1, CDH5, and MCAM. The downregulation of lymphatic endothelial markers such as FLT4, PROX1, and PDPN confirmed the identity of these cells as blood ECs rather than lymphatic ECs [42, 43]. Furthermore, the upregulation of COL4A1 and COL1A1 in ECs compared to iPSCs highlights the phenotypic transition toward a more mature endothelial state, characterized by enhanced ECM production [44, 45]. Interestingly, the expression of KDR, also known as VEGFR-2, remained low in both iPSCs and derived ECs, suggesting a potential regulatory mechanism during endothelial differentiation that warrants further investigation.

Pluripotency markers were downregulated compared to their iPSC origin. However, SOX2 continued to be upregulated compared to HUVECs. The exact role of SOX2 in EC biology remains under investigation, but emerging evidence suggests that it plays a crucial role in regulating EC behavior promoting angiogenesis, especially in response to environmental factors [49, 50]. Studies have shown that SOX2 expression is significantly elevated in the presence of VEGF, a potent promoter of EC proliferation and migration [51]. Moreover, recent findings indicate that excessive SOX2 signaling can alter the transcriptional landscape of brain ECs, leading to a disruption of the lumen in cerebral arteriovenous malformations [52]. However, further research is necessary to fully elucidate the mechanisms underlying SOX2-mediated angiogenesis and the implications for disease pathogenesis and treatment.

IPSC-derived VSMCs were generated through a defined monolayer culture yielding large quantities of proliferative, mature VSMCs. The efficacy of the differentiation process did not significantly differ between PiPSCs and UiPSCs yielding 96% and 94% of α-SMA positive cells, respectively. PiPSC-derived VSMCs and UiPSC-derived VSMC both exhibited a spindle shaped, fibrous morphology and demonstrated adequate contractility in response to carbachol. Transcriptomic analysis demonstrated pronounced upregulation of specific VSMC markers, such as ACTA2, CALD1, CNN1, TAGLN, and TPM2, confirming successful differentiation into VSMCs. The concurrent downregulation of ACTA1 and upregulation of ACTA2 in derived VSMCs further corroborates their vascular smooth muscle phenotype, given the preferential expression of ACTA1 in skeletal muscle and ACTA2 in vascular smooth muscle [37, 38]. The significant upregulation of COL6A1 in VSMCs indicates a shift toward a mature phenotype with enhanced ECM production, which is crucial for vascular tissue integrity and function [39–41].

Gene enrichment analysis of iPSCs derived from urine and PBMCs revealed distinct gene expression patterns, highlighting their unique molecular identities. In PiPSCs, enriched pathways related to cell cycle progression (e.g., E2F and Myc targets) suggested a higher potential for growth and proliferation. Conversely, UiPSCs showed increased expression in pathways related to cell–cell adhesion and tissue structure, such as apical junction and epithelial–mesenchymal transition, indicating changes in morphology and adhesion, with downregulated KRAS signaling implying reduced activity in growth pathways. PiPSC VSMCs showed heightened engagement of mitotic spindle and KRAS-mediated signaling pathways, suggesting roles in cell growth, survival, and differentiation, with activation of myogenesis aligning with smooth muscle cell phenotype. PiPSC ECs activated the P53 pathway, indicating roles in cell cycle control and DNA damage response, and upregulated angiogenesis, suggesting a pro-angiogenic phenotype. In contrast, UiPSC-derived ECs showed elevated expression of Myc targets and mitotic spindle pathways, indicating a growth-oriented state, with increased reliance on oxidative phosphorylation supporting active cellular processes.

The upregulation of key signaling pathways, such as KRAS, c-Myc, and E2F, in our cell lines holds, significant implications for their behavior and potential applications. Notably, the c-Myc pathway emerges as a pivotal player with a dual role, promoting both iPSC generation and malignant transformation factor, governing cellular proliferation and differentiation [53, 54]. The increased proliferation rates induced by c-Myc, coupled with the observation that it attenuates but does not eliminate differentiation capacity, emphasize its intricate role in balancing stem cell self-renewal and differentiation [55, 56]. However, as a proto-oncogene, c-Myc is a potent activator of carcinogenesis [57, 58].

The E2F transcription factor network regulates the expression of multiple genes associated with cell proliferation, encoding proteins that govern cell cycle progression, proliferation, apoptosis, differentiation, and development [59]. Notably, unlike c-Myc, it does not exhibit significant transforming activity [57].

While KRAS is recognized for its vital functions in maintaining stemness in certain stem cell types, its roles within pluripotent stem cells remain poorly elucidated [60]. This underscores the need for further investigation to better understand the influence of KRAS signaling on the self-renewal and differentiation propensity in iPSCs.

In summary, the upregulation of KRAS, c-Myc, and E2F pathways indicates a potential for enhanced proliferation, self-renewal, and altered differentiation capacities. However, the dual nature of these pathways as both drivers of normal cellular processes and potential contributors to malignancy underscores the need for careful monitoring and further investigation. Understanding the intricate balance of these pathways is crucial for optimizing the application of our cell lines in regenerative medicine and disease modeling, ensuring their safety and efficacy.

The study acknowledges several limitations. First, while iPSCs offer a versatile platform for studying cellular differentiation, the reprograming method itself may introduce biases that influence downstream analyses. Variability in reprograming efficiency and epigenetic memory from the somatic cell source could potentially affect the transcriptomic profiles observed in derived cell types. Second, while the differentiation protocols used in this study demonstrate robustness in generating VSMCs and ECs from both PiPSCs and UiPSCs, scalability for large-scale production remains a challenge. Optimization of differentiation efficiency and scalability is crucial for translating these findings into clinical applications, where consistent and scalable production of functional cells is essential. Lastly, while our findings provide valuable insights into the molecular characteristics of iPSC-derived VSMCs and ECs, further studies are needed to validate their relevance in clinical settings.

5. Conclusion

This study presents a comprehensive comparative analysis of functionally differentiated ECs and VSMCs derived from PiPSCs and UiPSCs, providing critical insights into the expression patterns and phenotypic transitions during differentiation. Our findings enhance the understanding of distinct molecular signatures in iPSCs from different sources and their progeny, contributing valuable knowledge for optimizing protocols to ensure functional and phenotypic fidelity, which is critical for therapeutic and modeling applications. Future studies are warranted to explore the efficacy of these cells in regenerative therapies for cardiovascular diseases or in modeling vascular pathologies to elucidate disease mechanisms and test novel therapeutics. Furthermore, given the concerns surrounding oncogenic risks associated with reprograming factors, there is a pressing need to develop strategies to mitigate these risks. Addressing these future directions will not only enhance the clinical relevance of iPSC-derived vascular cells but also contribute to advancing the safety and efficacy of iPSC-based therapies in clinical practice.

Disclosure

A preprint has previously been published [61]. The funding body played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

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