Long-term culture of skin biopsies: Maintenance of fibroblast production and competency of reprogramming

Long-term culture of skin biopsies: Maintenance of fibroblast production and competency of reprogramming | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Long-term culture of skin biopsies: Maintenance of fibroblast production and competency of reprogramming Sudiksha Rathan-Kumar, Michael A Ripperger, Grant M Westlake, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4651236/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Dec, 2025 Read the published version in FEBS Open Bio → Version 1 posted You are reading this latest preprint version Abstract Primary fibroblasts are a commonly used cell type used in a variety of experimental and therapeutic studies. Patient-derived skin biopsies are an accessible way to generate fibroblasts for use in various assays as well as reprogramming to iPSCs. To understand the potential of long-term skin biopsy culture, we cultured biopsy samples for 6 to 16 months and analyzed subsequent generation of fibroblasts. We found maintenance of morphology and physiology over time. Proliferation assays showed that older generations remained proliferative but at a decreased rate. mRNA analyses revealed transcriptional changes with long-term skin culture. Deep DNA sequencing did not reveal any large deletions or amplifications. Spontaneous DNA mutations seemed to be random and not enriched for any specific signaling pathways. Older fibroblasts generated after 16 months in culture retained competency for reprogramming into iPSCs. Our results support long term culture of skin to generate very large numbers of primary fibroblasts. These cells maintain their identity and integrity enabling the study of human disorders, particularly rare ones. Biological sciences/Stem cells/Mesenchymal stem cells Biological sciences/Biological techniques/Cytological techniques/Tissue culture Fibroblasts reprogramming skin culture Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Fibroblasts are one of the most abundant cells in the human body. First described in 1858, this cell type is present in a variety of organs including the skin, lungs, heart, and skeletal musculature 1 , 2 . Due to their tissue specific functions, they originate from various lineages, converging onto a fibroblast fate. They have been best studied in the skin, where dermal fibroblasts form a strong foundation through deposition of extracellular matrix, supporting keratinocytes, sweat glands, and hair follicles 1 . Fibroblast progenitors are of mesenchymal origin and generate dermal fibroblasts as well as dermal adipocytes 3 . Dermal fibroblasts play an important role in wound healing, initially interacting with immune cells during the inflammatory phase and improving the local immune response 4 , 5 . Fibroblasts also have a primary role during proliferative and skin remodeling, contributing to angiogenesis and secreting ECM molecules 6 , 7 . They also differentiate into myofibroblasts which regulate wound contraction and tissue remodeling to complete the wound healing process 8 . Due to this function, fibroblasts have also been highly studied regarding wound recovery and burn therapeutics 9 . Studies and clinical trials have shown that application of fibroblast growth factor (FGF) improves wound healing time and scar appearance 10 . Recent studies in animal models have demonstrated that transplantation of both allogeneic and autogenic fibroblasts onto wounds and ulcers improved wound healing 11 – 14 . Further studies utilized microcarriers and engineered sheets containing fibroblasts and other factors to promote wound healing 15 – 17 . Fibroblasts have also been used in skin replacement treatments for burn patients, with autologous cells being cultivated and transplanted onto the patient, leading to less complications during healing 18 . Another important utilization of fibroblasts is their ability to be reprogrammed to induced pluripotent stem cells (iPSC). Skin punch biopsies can be performed on healthy and individuals with disorders and primary fibroblast cell lines readily obtained from this biopsy 19 – 21 . Fibroblasts can be reprogrammed to iPSCs by exogenous expression of “Yamanaka factors” SOX2, cMYC, KLF4 , and OCT3/4 utilizing episomal vectors or non-integrative Sendai virus transduction 22 – 25 . A major application of this approach is to model genetic disorders as fibroblasts carry the same germline mutation in each patient and after production of iPSCs, can be differentiated to lineages of interest such as neurons, hepatocytes, and cardiomyocytes 26 , 27 . Human derived primary cells have then greatly expanded the field of biomedical research. In rare disease research, human derived cell models have proven invaluable in understanding disease mechanisms and developing therapeutics 28 , 29 . Recent legislature has also increased interest in the use of iPSCs in drug discovery and clinical trials 30 , 31 . However, for extremely rare diseases, due to the low number of patients, acquiring such cells has been a challenge for researchers and clinicians. Furthermore, the limiting effects of disease symptoms and the possible financial and medical burdens of travel might prevent participation and contribution of biopsy samples from certain patients and regions. Therefore, any obtained skin biopsy and the resulting fibroblasts are invaluable tools, especially for rare disorders. Previous research has demonstrated the applicability to cryopreserve biopsies for later culture 32 . However, there is a lack of investigation on the culturing potential of a single biopsy and the effects of extended culture on fibroblast formation and function. We now report a long-term study of biopsy-derived fibroblast cultures including the retained ability to reprogram to iPSCs from long-term fibroblast cultures. Our results indicate that patient-derived biopsies can produce very large numbers of fibroblasts for extended periods of time and these fibroblasts maintain genomic and physiological features. Our results address the ability of long-term cultures to produce patient-derived iPSCs and have broad applications in wound therapeutics and fibroblast physiology. MATERIALS AND METHODS Skin biopsies All skin biopsies were conducted under the approval of Health Sciences Review Committee #2 of the Institutional Review Board of Vanderbilt University Medical Center, IRB #080369. All research was performed in accordance with relevant guidelines/regulations including those in accordance with the Declaration of Helsinki. Informed consent/assent was obtained from each subject and a single 3 mm punch skin biopsy was obtained from the posterior arm of each subject. This chunk of tissue was dissected in half longitudinally cutting downwards from the exterior surface. Each half was then plated in a single well of a 6 well culture plate. A sterile glass slide cover was placed on the skin chunk. 3 mL of DMEM Complete (DMEM high glutamine, 10% FBS, Pen/Strep) was added to each well, completely submerging the biopsy and slide. Plates were incubated at 37C and 5% CO 2 , gently aspirating and changing media twice a week. After 1–3 weeks, fibroblast appearing cells began to migrate out. When confluent, skin chunks were moved to a new 6-well plate. Fibroblasts from the original well were then passaged using 0.1% Trypsin-EDTA and plated on a 10 cm petri dish with DMEM Complete for further growth and expansion before cryopreservation. For storage, cells were trypsinized and resuspended in fibroblast freezing media (50% DMEM, 40% FBS, 10% DMSO). The cell solution was then aliquoted into multiple cryotubes for each generation, frozen overnight at -80C and then moved to liquid nitrogen for long-term storage. Initial fibroblasts grown from the skin chunk were denoted as the first generation (G1) and subsequent chunk passaging and resulting fibroblast cultures G2-G16. Patient Selection We focused on a punch biopsy from a male patient with Hikeshi Associated Leukodystrophy (HAL), a devastating neurological disorder caused by homozygous loss of function mutations in the HIKESHI gene 33 – 35 . Control lines were obtained from first degree relatives with a heterozygous mutation of the HIKESHI gene. We used male and female first-degree relatives as control lines. The genotype of all lines was confirmed by DNA sequencing and further validated by immunostaining for HIKESHI protein. Immunohistochemistry Skin biopsies were removed from culture plate and fixed in 4% paraformaldehyde (PFA) for a minimum of 24 hours. The biopsies were then transferred into 70% Ethanol and submitted to the Vanderbilt Translational Pathology Shared Resource. Fixed tissue was embedded into paraffin and cut into 5 um thick sections. One set of slides was stained with hematoxylin and eosin (H&E). The H&E-stained slides were submitted to the Vanderbilt Digital Histology Shared Resource and imaged with the Leica SCN400 Slide Scanner (Leica Biosystems) at 40x magnification. FFPE Slide Immunofluorescence Tissue sections were deparaffinized and rehydrated by immersion in xylene and a series of ethanol dilutions. Antigen retrieval was performed utilizing heated citrate buffer pH 6. Slides were incubated with blocking buffer for 1 hour at room temperature. Primary antibodies (Table 1 ) were diluted in the blocking buffer and 100 uL added to each well in a humidified chamber at 4C overnight. Secondary antibodies were diluted in blocking buffer and incubated at room temperature for 2 hours. Slides were then washed twice with 1X PBS and mounted using Prolong Antifade DAPI mounting medium (Invitrogen) and allowed to dry overnight before imaging on the EVOS FL Auto microscope. Table 1 Primary Antibodies used, source, and dilution. Antibody Antibody Registry ID Usage CC3 AB_2341188 1:500 FSP AB_10000870 1:500 Ki67 AB_302459 1:250 Ki67 AB_ 10854564 1:400 PCNA AB_ 2160343 1:500 Nanog AB_10559205 1:200 Oct4 AB_823583 1:200 SSEA3 AB_177628 1:100 SSEA4 AB_528477 1:200 TRA-1-60 AB_2119183 1:200 Vimentin AB_10695459 1:500 Chambered Slide Immunofluorescence Ibidi 8-well chambered slides (Ibidi 80841) were coated with poly-L-lysine (Sigma Aldrich P4707) for 20 minutes. Poly-L-lysine was aspirated, and slides were washed twice with dH2O and air dried for 2 hours. Fibroblasts were harvested from a confluent T25 flask using 0.25% Trypsin-EDTA solution (Gibco 25200-056) for 7 minutes at 37C before spun down at 300xG for 5 minutes and the pellet was resuspended in 1mL DMEM Complete. A Countess Cell Counter was used cells resuspended in DMEM Complete, plating 2000 cells into each well of the slide. Slides were incubated overnight at 37C. Media was aspirated, and slides were washed with 1X PBS before proceeding with antibody staining as above. Cell Proliferation Counting Fibroblasts were plated on 8-well chambered slides at 2,500 cells per well. Cells were fixed and stained for expression of Ki67 and imaged on the EVOS FL Auto microscope at 10x magnification. EVOS Software captured 15 randomized images each from 4 wells. The captured images were analyzed and the total number of DAPI nuclei and Ki67 positive cells were counted. Cell counts were averaged for the well and each well was denoted as a data point for statistical analysis and graphing. Proliferation Assay A proliferation assay was conducted on fibroblasts over 7-day period. Fibroblasts were harvested from a confluent T25 flask using 0.25% Trypsin-EDTA solution (Gibco 25200-056) for 7 minutes at 37C. Fibroblasts were spun down, and pellet was resuspended in 1mL DMEM Complete. Cells were counted on the Nexcelom Cellometer Auto T4 Cell Counter and resuspended in DMEM Complete at 10,000 cells/mL. 100 uL of cell solution was added to 8 wells per genotype of 8 96-well plates (1 for each day) for 1,000 cells per well. Plates were incubated at 37C. A Thermofisher CyQuant Direct Cell Proliferation Assay (C35011) was utilized for analysis. For Day 0 to Day 7, the staining solution was prepared per the kit’s instructions and 100 uL was added per well. Plates were incubated for 1 hour at 37C. Plates were then read at 485/520nm by the Biotek Synergy HT plate reader. Data and statistics were compiled using Biotek Gen5 and Prism GraphPad. RNA Sequencing Fibroblasts were harvested and resuspended as above. RNA was extracted using the Qiagen RNAeasy Kit (Qiagen 74104) and Qiagen RNA-free DNase kit (Qiagen 79254). Samples were quantified using a NanoDrop and submitted to Vanderbilt Technologies for Advanced Genomics core laboratory. Paired-end reads were obtained from Illumina NovaSeq6000 PE150 Sequencing. RNA sequence processing was performed with Pyrpipe v0.0.5. First sequence adapters from the reads were trimmed with Trimgalore v0.6.6 and aligned with STAR v2.7.7a to the Genome Reference Consortium Human Build 38 patch release 14 (GRCh38.p14). Assembly of alignments was done with StringTie v2.14 and the GRCh38.p14 NCBI RefSeq annotation assembly (GCF_000001405.40). Gene abundances were estimated using StringTie. Gene and transcript read counts were extracted for each sample with prepDE.py provided by JHU CCB. Pydeseq2 v0.3.5 was used to perform single and multiple factor differential expression analysis among Generations 1 and 6 for both cell lines. Each paired read was treated as a replicate. Genes with adjusted p-values by the Benjamini and Hochberg method under or equal to 0.001 and log fold change greater than 1 or less than − 1 were selected for Gene Ontology Enrichment Analysis (GOEA) using Goatools (v1.3.2) 36 . All human genes from Gencode annotations release 39 (GRCh38.p13) served as the background population set. Benjamini-Hochberg p-values were obtained for each Gene Ontology biological process, cellular component, and molecular function pathway. Upregulated and downregulated genes were analyzed separately. Gene associations were taken from the human Gene Ontology 2023 library. Whole Genome Sequencing Fibroblasts were harvested and resuspended as above. DNA was extracted using the DNAeasy Kit (Qiagen 69504). Samples were quantified using a NanoDrop. Samples were submitted to CD Genomics for low-pass whole genome sequencing and analysis. Samples were run through a quality test then utilized to create a construct library. The generated library was sequenced using the Illumina HiSeq sequencing platform producing raw data. The raw data was processed to filter out various reads including low quality and redundant reads. The sequencing quality score is determined, and the distribution of base content is calculated from the raw data. The reads were then aligned with a reference genome and Copy Number Variations (CNV) determined. Deletions and duplications were identified and annotated with gene names or Ensembl IDs. Chromosome diagrams were generated for each genotype depicting the CNVs. Statistics were conducted throughout the process for sequencing reads, alignment results and more. GOEA was performed in the same manner as with gene expression using DNA duplications and deletions from Generation 16 fibroblasts. Both duplications and deletions were considered a change to the gene and used as the study population. Changes already present in Generation 1 were removed from this set. A p-value of 0.001 or less was used to identify modified pathways. Fibroblast Reprogramming Fibroblasts were reprogrammed using the CytoTune-iPS 2.0 Reprogramming Kit (Invitrogen A16517). Fibroblasts were plated at 2x10 5 cells per well of a Matrigel-coated 6-well plate. Cells were fed with DMEM Complete. The following day (Day 0), 1 well was disassociated from the plate and the cell number was counted. The volume of virus required was calculated using the formula: $$Vol of virus \left(ul\right)=\frac{MOI x cells/ml}{\begin{array}{c}\frac{CIU}{ml}x\frac{{10}^{-3}ml}{ul}\\ \end{array}}$$ . Viruses hKOS and cMYC had a MOI of 5 and KLF4 a MOI of 3. Virus was added to DMEM complete w/o PenStrep, adding 1 mL per well of cells. The following day (D1), the virus solution was carefully removed, and the cells were fed with DMEM Complete, changing the media every day from Day 1 to Day 6. On Day 7, the cells were disassociated with 0.25% Trypsin and counted. They were plated on Matrigel-coated 6 well plates with densities of 5x10 4 , 7.5X10 4 and 1x10 5 cells per well. The cells were moved to Essential 8 Medium (Gibco A15169-01) with Supplement (Gibco A15171-01). Cells were then grown in E8 Medium, feeding every other day from Day 7 to Day 21. Cells were then moved to mTesR1 media (StemCell Technologies #85857). Individual colonies were identified and picked by scraping, colonies were transferred to a Matrigel-coated 48 well plate and grown on mTeSR1. Colonies were passaged over multiple generations and then fixed with 4% PFA for 30 minutes at room temperature. Immunofluorescence on colonies was done as above. Data Availability RNA Sequencing data was deposited in Gene Expression Omnibus (GEO) under accession GSE271347. RESULTS To generate primary dermal fibroblast cultures, for years we have been obtaining skin biopsies from patients with rare neurological disorders. The fibroblasts are used for experiments and for reprogramming to iPSCs. Given the very rare nature of some disorders we have studied, the initial skin biopsy from a patient with Hikeshi Associated Leukodystrophy (HAL, OMIM # 616881) was propagated further after initial fibroblasts were collected. We had previously observed the potential of human skin chunks to continue to generate fibroblasts when the biopsy was placed in a new well. As patients with HAL can decompensate and even die following fever and infection, we strove to limit exposure to any invasive procedures including skin biopsy. We continued to propagate this individual chunk of skin for greater than 15 months. We noted the continued generation of fibroblasts, even from latter generations. We hypothesized that long-term culture of the skin chunk did not impede fibroblast identity and function including cell morphology, gene expression, and ability to be reprogrammed to iPSCs. The HAL patient biopsy (HIKHom-1) was propagated for 16 generations with many tubes of fibroblasts frozen down at each passage upon transfer of the skin chunk to a new well. Control cultures derived from first-degree relatives with heterozygous HIKESHI mutations (HIKHet-2) were also propagated, summarized in Table 2 . Table 2 Skin Biopsies used and Fibroblast Generations Post Biopsy. Label Genotype Generation Days since biopsy HetG1 HIKHet-2 1 9 days HetG6 HIKHet-2 6 159 days HomG1 HIKHOM-1 1 71 days HomG6 HIKHOM-1 6 190 days HomG16 HIKHOM-1 16 473 days After final generation of fibroblasts, the HIKHOM-1 and HIKHet-2 skin chunks were harvested, fixed, and sectioned. H&E stains of HIKHOM-1 and HIKHet-2 were compared to two unrelated control skin biopsies harvested after one generation. Likely due to age (G16 versus G6) the HIKHOM-1 skin biopsy appeared thinner and more damaged compared to control skin biopsies. The HIKHet-2 biopsy was also thinner and appeared more damaged than the controls (Fig. 1 ). All fibroblasts regardless of age, generation, and genotype exhibited the characteristic spindle morphology (Fig. 2 ), swirled patterns of growth, and contact inhibition when cultures were confluent 41 . Therefore, the cells seemed to retain fibroblast lineage and morphology over various generations, suggesting that long-term biopsy culture does not affect the cell type or morphology. As we hypothesized that resident progenitor cells within each piece of skin remained proliferative and capable of producing fibroblasts, we used immunofluorescence for expression of Fibroblast-Specific Protein 1 (FSP1), a member of the S100 calcium-binding protein superfamily 37 . FSP1 is present in fibroblasts and absent in epithelial, mesangial, and embryonic endoderm cells 38 . Cells were also stained for the cytoskeletal protein Vimentin, which has also been frequently used as a fibroblast marker 39 , 40 (Fig. 2 ). The staining displayed that the cells had both the spindle-shaped body associated with inactive fibroblasts and the larger, more stellate bodies of active fibroblasts. Cell Proliferation and Death We thawed fibroblasts from different generations and genotypes and measured proliferation over 7 days (Fig. 3 A). There were no significant changes in proliferation between HIKHet-2 G1 and HIKHet-2 G6 over 7 days. However, there was a significant decrease in proliferation between HIKHOM-1 G1 and HIKHOM-1 G6 Day 6 onwards (Fig. 3 A). Furthermore, there was a significant decrease in proliferation in HIKHOM-1 G16 compared to both HIKHOM-1 G1 and HIKHOM-1 G6 from Day 4 onwards. It was also observed that the HIKHOM-1 cell lines overall had a higher proliferative rate than HIKHet-2 cells. To determine the percentage of proliferating cells, cells were fixed and stained for a proliferation marker, Ki67 42 (Fig. 3 B). Cell counts indicated that the percentage of Ki67 expressing cells decreased over generations. There was a downward trend between Gen1 and Gen6 of both HIKHet-2 and HIKHOM-1 genotypes, however this decrease was not significant. There was a significant decrease between HIKHOM-1 G1 and HIKHOM-1 G16, suggesting that long-term culture slows but does not halt proliferation by 473 days. Similar to the proliferative assay, the HOM genotype displayed a higher percentage of Ki67 positive cells across all generations when compared to the Het cells. To analyze the presence of proliferative cells in the biopsy samples after long-term culture, the biopsies were ultimately fixed, paraffin-embedded, and immunostained for proliferative markers. Immunofluorescence staining indicates a lack of Ki67 positive and PCNA positive cells in HIKHOM-1, HIKHet-2, and unrelated control skin biopsies (data not shown). Validity of this assay was confirmed utilizing multiple antibodies and positive control tissue. Cells were also fixed and stained for the apoptosis marker, Cleaved Caspase 3 (CC3) 43 . Fibroblasts were negative for CC3 indicating minimal if any apoptotic cell death (data not shown). Antibody validity was confirmed utilizing positive control cells. Gene Expression and Genomic Integrity To determine if there were appreciable differences in gene expression, RNA was obtained from proliferating fibroblast lines from different generations and genotypes. RNA sequencing was conducted, and we observed significant upregulation and downregulation of various genes between HIKHOM-1 G1 and G16 (Fig. 4 ). Various genes such as HAPLN1, LHX , and SDK2 were highly upregulated and SMO, C3 , and EMILIN2 were highly downregulated. Gene enrichment analysis between HIKHOM- 1 G1 and G16 indicated that a variety of pathways were changed. Pathways relating to the extracellular matrix, ion channel activity, and transmembrane transport were upregulated. Pathways relating to the regulation of the immune system, response to stimulus, and cell surface receptor signaling were downregulated. Analysis between HIKHOM-1 G1 and G6 as well as HIKHet-2 G1 and G6 also displayed changes in expression and gene enrichment, but substantially fewer changes to expression and pathways were observed implying long-term culture amplified transcriptional changes (Supplementary Fig. 1). As prolonged time in culture could predispose cells to genomic instability and acquisition of mutations, we processed DNA from proliferating fibroblast lines from different generations and genotypes for low-pass whole genome sequencing. This allowed us to assess chromosomal stability of long-term culture, and to detect any large DNA sequence gains or losses. Chromosomal maps indicate that there were no significant chromosomal gains or losses that developed over generations (Supplementary Fig. 2). Further enrichment analysis determined that between HIKHom1 G1 and G16, only a single pathway was significantly changed. A deletion in Chromosome 22 affected genes in the APOBEC3 cluster including APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D and APOBEC3F . This resulted in potential reductions in deaminase activity and negative regulation of viral processes 44 – 47 . Despite these genomic changes, the cells displayed no increased susceptibility to contamination. The genomic analysis also indicated that mutations occurred at random and no specific pathway, such as cancer-related pathways were targeted. Reprogramming Competence To determine if prolonged propagation of skin biopsies could produce fibroblasts that are competent for reprogramming to iPSC, HIKHet-2 G1 and HIKHOM-1 G16 fibroblasts were reprogrammed using Sendai virus containing Yamanaka factors. By Day 16, colonies of round shaped cells were evident as well as surrounding cells maintaining a typical fibroblast morphology. On Day 21 since transduction with Sendai viruses, emerging iPSC-like colonies were manually picked and replated. These were grown out for a few more days before being imaged. The individual colonies were flat and consistent with iPSC morphology (Fig. 5 A), consisting of small, round cells with high nuclear/cytoplasmic ration with prominent nucleoli 48 . To provide further evidence that HIKHOM-1 G16 fibroblasts were competent for reprogramming, individual colonies were immunostained for pluripotency markers Nanog, Oct4, TRA-1-60, SSEA3, and SSEA4 49,50 . All lines stained positive for all markers (Fig. 5 B), indicating pluripotent status and successful reprogramming. DISCUSSION This study demonstrates the long-term viability and reprogramming competency of skin biopsy derived fibroblast cultures. A prolonged 16-month culture of a single 3 mm skin biopsy produced 16 generations of fibroblasts amounting to potentially billions of cells. These fibroblasts have been utilized for a variety of experiments including immunofluorescence, immunoblotting, RNA and DNA sequencing, and iPSC reprogramming. We observed retention of fibroblast morphology and marker expression, further supporting the long-term maintenance of the cellular identity. Proliferation assays showed that later generations have decreased proliferative capacity compared to the first generation of fibroblasts. HIKHOM1 fibroblasts did show an overall higher level of proliferation compared to HIKHet2 cells which could be due to genotype as well as age differences 51 . At the end of the prolonged culture period, we did not observe proliferative cells in the fixed and sectioned skin biopsies. Due to the small number of cells visible in the biopsy, this could be due to the tissue sections lacking proliferative cells. Previous studies have also shown that extended formalin-fixation can reduce the Ki67 staining 52 . Cultured fibroblasts were negative for cleaved caspase 3 (CC3) cells indicating minimal if any apoptotic cell death. As plated cells were not confluent, the fibroblasts appear to be continuously proliferative but exhibited contact inhibition 53 , 54 . As the cultured fibroblasts were passaged or frozen upon confluency, this could result in a lack of apoptotic pressure on the cells. RNA and DNA sequencing demonstrate the overall maintenance of genomic integrity over long-term culture. There were no chromosomal abnormalities over fibroblast generations. The few acquired mutations seem to have occurred at random and did not appear to specifically target a pathway or affect the physiology and culture potential of the cells. Studies on other cell types such as mesenchymal stem cells have demonstrated that long-term culture can result in random mutation accumulation with oxidative stress of culture conditions playing a primary role 55 – 57 . Further experiments would determine if regulation of culture conditions to reduce oxidative stress will reduce mutation frequency in long-term skin-biopsy culture. Extensive changes in expression were observed between the first and last generation, similar to studies observing long-term culture of mesenchymal stem cells 58 . The resulting protein changes and functional differences caused by these expression changes will require further study. Successful reprogramming of G16 fibroblasts indicates that reprogramming competence was not altered over long-term culture of fibroblasts. Due to the DNA and RNA changes, reprogrammed stem cells would have to be further sequenced and propagated to determine that the apparent randomly acquired changes do not affect stem cell physiology and differentiation potential. If long-term fibroblast culture is utilized in clinical applications, both directly and for reprogramming, cells will have to be frequently sequenced to ensure that no harmful mutations have been acquired that may reduce therapeutic potential or cause a host response. In this report we determined the prolonged capacity and robustness of skin biopsies to continuously produce many generations of primary fibroblasts. Especially for rare genetic diseases, these findings have important implications in primary cell culture and in vitro translational research. Declarations Funding Sources: Institutional Funding from Vanderbilt University Medical Center and University of Colorado Anschutz Medical Campus Conflict of Interest: The authors declare no conflicts of interest Author Contribution SRK and KCE conceived and designed the study. SRK, KCE, and GMW performed experiments as well as data collection. SRK, MAR, and KCE analyzed the data and interpreted the results. SRK and KCE wrote the manuscript. All authors reviewed the results and approved the final version of the manuscript. Acknowledgement We thank the patient with HAL and their family for allowing us to obtain skin biopsies. We also thank our funding sources who wish to remain anonymous. We thank Brittany Parker Short and Tenhir Iyer for technical assistance. Whole slide imaging was performed in the Digital Histology Shared Resource at Vanderbilt University Medical Center (www.mc.vanderbilt.edu/dhsr). 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Reprogramming human dermal fibroblast into induced pluripotent stem cells using non-integrative Sendai virus for transduction. Takahashi K, Tanabe K, Ohnuki M, et al. Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell. 2007;131(5):861–872. doi: 10.1016/J.CELL.2007.11.019 Lyra-Leite DM, Gutiérrez-Gutiérrez Ó, Wang M, Zhou Y, Cyganek L, Burridge PW. A review of protocols for human iPSC culture, cardiac differentiation, subtype-specification, maturation, and direct reprogramming. STAR Protoc. 2022;3(3):101560. doi: 10.1016/J.XPRO.2022.101560 Sharma A, Sances S, Workman MJ, Svendsen CN. Multi-lineage Human iPSC-Derived Platforms for Disease Modeling and Drug Discovery. Cell Stem Cell. 2020;26(3):309–329. doi: 10.1016/J.STEM.2020.02.011 Anderson RH, Francis KR. Modeling Rare Diseases with Induced Pluripotent Stem Cell Technology. Mol Cell Probes. 2018;40:52. doi: 10.1016/J.MCP.2018.01.001 Saito MK, Osawa M, Tsuchida N, et al. A disease-specific iPS cell resource for studying rare and intractable diseases. Inflamm Regen. 2023;43(1). doi: 10.1186/S41232-023-00294-2 Kim JY, Nam Y, Rim YA, Ju JH. Review of the Current Trends in Clinical Trials Involving Induced Pluripotent Stem Cells. Stem Cell Rev Rep. 2022;18(1):142. doi: 10.1007/S12015-021-10262-3 Zushin PJH, Mukherjee S, Wu JC. FDA Modernization Act 2.0: transitioning beyond animal models with human cells, organoids, and AI/ML-based approaches. J Clin Invest. 2023;133(21). doi: 10.1172/JCI175824 F Gray RG, Ryan D, Green A. The cryopreservation of skin biopsies-a technique for reducing workload in a cell culture laboratory. Original Article Ann Clin Biochem. 1995;32:190–192. Helman G, Zerem A, Almad A, et al. Further Delineation of the Clinical and Pathologic Features of HIKESHI-Related Hypomyelinating Leukodystrophy. Pediatr Neurol. 2021;121:11–19. doi: 10.1016/J.PEDIATRNEUROL.2021.04.014 Vasilescu C, Isohanni P, Palomäki M, Pihko H, Suomalainen A, Carroll CJ. Absence of Hikeshi, a nuclear transporter for heat-shock protein HSP70, causes infantile hypomyelinating leukoencephalopathy. European Journal of Human Genetics. 2017;25(3):366–370. doi: 10.1038/ejhg.2016.189 Edvardson S, Kose S, Jalas C, et al. Leukoencephalopathy and early death associated with an Ashkenazi-Jewish founder mutation in the Hikeshi gene. J Med Genet. 2016;53(2):132–137. doi: 10.1136/jmedgenet-2015-103232 Klopfenstein D V., Zhang L, Pedersen BS, et al. GOATOOLS: A Python library for Gene Ontology analyses. Scientific Reports 2018 8:1 . 2018;8(1):1–17. doi: 10.1038/s41598-018-28948-z Fanò G, Biocca S, Fulle S, Mariggiò MA, Belia S, Calissano P. The S-100: A protein family in search of a function. Prog Neurobiol. 1995;46(1):71–82. doi: 10.1016/0301-0082(94)00062-M Strutz F, Okada H, Lo CW, et al. Identification and characterization of a fibroblast marker: FSP1. J Cell Biol. 1995;130(2):393. doi: 10.1083/JCB.130.2.393 Goodpaster T, Legesse-Miller A, Hameed MR, Aisner SC, Randolph-Habecker J, Coller HA. An Immunohistochemical Method for Identifying Fibroblasts in Formalin-fixed, Paraffin-embedded Tissue. Journal of Histochemistry and Cytochemistry. 2008;56(4):347. doi: 10.1369/JHC.7A7287.2007 Ostrowska-Podhorodecka Z, Ding I, Norouzi M, McCulloch CA. Impact of Vimentin on Regulation of Cell Signaling and Matrix Remodeling. Front Cell Dev Biol. 2022;10:869069. doi: 10.3389/FCELL.2022.869069/BIBTEX Fernandez-Madrid F, Noonan S, Riddle J. The “spindle-shaped” body in fibroblasts: intracellular collagen fibrils. J Anat. 1981;132(Pt 2):157. Scholzen T, Gerdes J. The Ki-67 Protein: From the Known and the Unknown. Published online 2000. doi: 10.1002/(SICI)1097-4652(200003)182:3 Porter AG, Jänicke RU. Emerging roles of caspase-3 in apoptosis. Cell death and differentiation. 1999;6(2):99–104. doi: 10.1038/SJ.CDD.4400476 Refsland EW, Harris RS. The APOBEC3 Family of Retroelement Restriction Factors. Curr Top Microbiol Immunol. 2013;371:1. doi: 10.1007/978-3-642-37765-5_1 Chiu YL, Greene WC. The APOBEC3 cytidine deaminases: an innate defensive network opposing exogenous retroviruses and endogenous retroelements. Annu Rev Immunol. 2008;26:317–353. doi: 10.1146/ANNUREV.IMMUNOL.26.021607.090350 Jarmuz A, Chester A, Bayliss J, et al. An anthropoid-specific locus of orphan C to U RNA-editing enzymes on chromosome 22. Genomics. 2002;79(3):285–296. doi: 10.1006/geno.2002.6718 Uriu K, Kosugi Y, Ito J, Sato K. The Battle between Retroviruses and APOBEC3 Genes: Its Past and Present. Viruses. 2021;13(1). doi: 10.3390/V13010124 Wakui T, Matsumoto T, Matsubara K, Kawasaki T, Yamaguchi H, Akutsu H. Method for evaluation of human induced pluripotent stem cell quality using image analysis based on the biological morphology of cells. Journal of Medical Imaging. 2017;4(4):1. doi: 10.1117/1.JMI.4.4.044003 Zhao W, Ji X, Zhang F, Li L, Ma L. Embryonic Stem Cell Markers. Molecules. 2012;17(6):6196. doi: 10.3390/MOLECULES17066196 Baghbaderani BA, Syama A, Sivapatham R, et al. Detailed Characterization of Human Induced Pluripotent Stem Cells Manufactured for Therapeutic Applications. Stem Cell Rev. 2016;12(4):394. doi: 10.1007/S12015-016-9662-8 Cockey GH, Boughman JA, Harris EL, Hassell TM. Genetic control of variation in human gingival fibroblast proliferation rate. In Vitro Cellular & Developmental Biology. 1989;25(3):255–258. doi: 10.1007/BF02628463/METRICS Hitchman E, Hodgkinson C, Roberts D, et al. Effect of prolonged formalin fixation on immunohistochemical staining for the proliferation marker Ki67. Histopathology. 2011;59(6):1261–1263. doi: 10.1111/J.1365-2559.2011.03974.X Abercrombie M, Lamont M, Stephenson EM. The monolayering in tissue culture of fibroblasts from different sources. Proc R Soc Lond B Biol Sci. 1968;170(1021):349–360. doi: 10.1098/RSPB.1968.0044 Ribatti D. A revisited concept: Contact inhibition of growth. From cell biology to malignancy. Exp Cell Res. 2017;359(1):17–19. doi: 10.1016/J.YEXCR.2017.06.012 Thompson O, von Meyenn F, Hewitt Z, et al. Low rates of mutation in clinical grade human pluripotent stem cells under different culture conditions. Nature Communications. 2020;11(1). doi: 10.1038/S41467-020-15271-3 Kuijk E, Jager M, van der Roest B, et al. The mutational impact of culturing human pluripotent and adult stem cells. Nature Communications. 2020;11(1). doi: 10.1038/S41467-020-16323-4 Wang Y, Zhang Z, Chi Y, et al. Long-term cultured mesenchymal stem cells frequently develop genomic mutations but do not undergo malignant transformation. Cell Death & Disease 2013 4:12 . 2013;4(12):e950-e950. doi: 10.1038/cddis.2013.480 Wang S, Wang Z, Su H, et al. Effects of long-term culture on the biological characteristics and RNA profiles of human bone-marrow-derived mesenchymal stem cells. Molecular Therapy Nucleic Acids. 2021;26:557–574. doi: 10.1016/j.omtn.2021.08.013 Additional Declarations No competing interests reported. Supplementary Files SuppleFigures.pdf Cite Share Download PDF Status: Published Journal Publication published 18 Dec, 2025 Read the published version in FEBS Open Bio → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4651236","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":329101406,"identity":"c1b3b4fd-285a-474d-82be-9faf4c57eea1","order_by":0,"name":"Sudiksha Rathan-Kumar","email":"","orcid":"","institution":"Vanderbilt University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Sudiksha","middleName":"","lastName":"Rathan-Kumar","suffix":""},{"id":329101407,"identity":"7aed3559-8fa5-41bc-b336-206de5668762","order_by":1,"name":"Michael A Ripperger","email":"","orcid":"","institution":"Vanderbilt University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"A","lastName":"Ripperger","suffix":""},{"id":329101408,"identity":"595ac23a-663e-4f22-a000-6c47f982119f","order_by":2,"name":"Grant M Westlake","email":"","orcid":"","institution":"Vanderbilt University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Grant","middleName":"M","lastName":"Westlake","suffix":""},{"id":329101409,"identity":"350f8f0d-a503-4d4b-a113-3dce39236e5e","order_by":3,"name":"Kevin C Ess","email":"data:image/png;base64,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","orcid":"","institution":"University of Colorado Anschutz Medical Campus","correspondingAuthor":true,"prefix":"","firstName":"Kevin","middleName":"C","lastName":"Ess","suffix":""}],"badges":[],"createdAt":"2024-06-28 00:36:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4651236/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4651236/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1002/2211-5463.70136","type":"published","date":"2025-12-19T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61194262,"identity":"94f0b126-19ca-4d49-8fae-b3b7eeaa3bca","added_by":"auto","created_at":"2024-07-26 21:08:57","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":199853,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSkin biopsies display deterioration over time in culture: \u003c/strong\u003eSkin biopsies from HIKHOM-1, HIKHet-2 and unrelated controls, CH1-CON and CH2-CON were fixed, paraffin embedded, and H\u0026amp;E stained. Slides were imaged on Aperio Versa 200 Bright Field microscope at 40x magnification.\u003cstrong\u003e \u003c/strong\u003eAs evidenced by vacuolar changes and issue integrity, long-term culture resulted in deterioration of the HIKHOM-1 and HIKHet-2 skin chunks compared to the control pieces of skin.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4651236/v1/1512e75ae8387b7a8d45f8b0.jpg"},{"id":61194264,"identity":"5053f355-952e-416f-8ad8-c49a0213a302","added_by":"auto","created_at":"2024-07-26 21:08:57","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":99879,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFibroblasts retain spindle-morphology and stain for markers\u003c/strong\u003e. (A) Phase contrast imaging of fibroblasts displays characteristic spindle-shape, indicating maintenance of morphology, scale bar: 100 um. (B) Cells were fixed and stained for FSP and Vimentin that were tested positive in all lines. Scale bar: 100 um.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4651236/v1/dce8f4dccb49725a523c51a9.jpg"},{"id":61194265,"identity":"3666ffa9-d92b-4f19-a5e5-fe1f59059f67","added_by":"auto","created_at":"2024-07-26 21:08:57","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":70728,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLater generations of fibroblasts are proliferative but at a decreased rate\u003c/strong\u003e. (A) Cells were plated and grown over 7 days. Cell numbers were measured each day utilizing CyQuant Direct Cell Proliferation Assay. Analyses were conducted using repeated- measures ANOVA analysis for 8 replicate wells per day. HIKHet-2 Gen 6 did not have any significant changes compared to HIKHet-2 Gen 1. (B) Compared to HIKHOM-1 Gen1, HIKHOM-1 Gen 6 and Gen 16 proliferation was significantly decreased by Day 6 and 4 respectively (p\u0026lt;0.0001) (C) Fibroblasts were cultured, fixed, immunostained for Ki67, and imaged at 10x magnification. Ki67+ve cells were counted and compared using one-way ANOVA analysis. Compared to HIKHOM-1 Gen1, HIKHOM-1 Gen 16 had significantly decreased Ki67+ve cells (4 technical replicates, p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4651236/v1/a45f2872d6797100afa03860.jpg"},{"id":61194266,"identity":"afb7b739-54b8-4a9c-b57d-985c25bc0f38","added_by":"auto","created_at":"2024-07-26 21:08:57","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":91817,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRNA Sequencing shows significant transcriptional changes between HIKHOM-1 Gen1 and Gen16: \u003c/strong\u003eRNA was extracted from\u003cstrong\u003e \u003c/strong\u003ecell lines and sequenced on the Illumina NovaSeq6000 platform. Data was processed and plotted in a volcano plot with transcripts greater than a 2-fold change and p\u0026lt;0.001 deemed biologically and statistically significant.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4651236/v1/69f929fb3d08d5d664884f98.jpg"},{"id":61194267,"identity":"7fb49123-71b1-46de-853f-a1d3da122381","added_by":"auto","created_at":"2024-07-26 21:08:57","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":236733,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHIKHOM-1 Gen16 fibroblasts were successfully reprogrammed into iPSCs\u003c/strong\u003e. (A) Reprogrammed HIKHOM-1 Gen16 and HIKHet-2 Gen1 fibroblasts imaged 20 days post-reprogramming (phase contrast). Cells were observed to change from fibroblast spindle-morphology to iPSC appearing colonies. Scale bar: 100 um. (B) Reprogrammed iPSCs for HIKHOM-1 Gen16 were fixed and fluorescently stained for pluripotency markers TRA-1-60, OCT4, NANOG, SSEA3, and SSEA4. Scale bar: 100 um.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4651236/v1/f53855b6dcaafd0b86a5a868.jpg"},{"id":102874794,"identity":"a2798493-1fde-4e0c-8578-7cd681dc254e","added_by":"auto","created_at":"2026-02-17 19:21:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1544585,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4651236/v1/088f2b69-c0ce-4baf-9150-ad62112f77b7.pdf"},{"id":61194263,"identity":"79acb0c6-3e32-46e5-a7f5-4ef4ca7e8a79","added_by":"auto","created_at":"2024-07-26 21:08:57","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":433993,"visible":true,"origin":"","legend":"","description":"","filename":"SuppleFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4651236/v1/9dc4232f51cbb194c5b00c10.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Long-term culture of skin biopsies: Maintenance of fibroblast production and competency of reprogramming","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eFibroblasts are one of the most abundant cells in the human body. First described in 1858, this cell type is present in a variety of organs including the skin, lungs, heart, and skeletal musculature\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Due to their tissue specific functions, they originate from various lineages, converging onto a fibroblast fate. They have been best studied in the skin, where dermal fibroblasts form a strong foundation through deposition of extracellular matrix, supporting keratinocytes, sweat glands, and hair follicles\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Fibroblast progenitors are of mesenchymal origin and generate dermal fibroblasts as well as dermal adipocytes\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDermal fibroblasts play an important role in wound healing, initially interacting with immune cells during the inflammatory phase and improving the local immune response\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Fibroblasts also have a primary role during proliferative and skin remodeling, contributing to angiogenesis and secreting ECM molecules\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. They also differentiate into myofibroblasts which regulate wound contraction and tissue remodeling to complete the wound healing process\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDue to this function, fibroblasts have also been highly studied regarding wound recovery and burn therapeutics\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Studies and clinical trials have shown that application of fibroblast growth factor (FGF) improves wound healing time and scar appearance\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Recent studies in animal models have demonstrated that transplantation of both allogeneic and autogenic fibroblasts onto wounds and ulcers improved wound healing\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Further studies utilized microcarriers and engineered sheets containing fibroblasts and other factors to promote wound healing\u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Fibroblasts have also been used in skin replacement treatments for burn patients, with autologous cells being cultivated and transplanted onto the patient, leading to less complications during healing\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAnother important utilization of fibroblasts is their ability to be reprogrammed to induced pluripotent stem cells (iPSC). Skin punch biopsies can be performed on healthy and individuals with disorders and primary fibroblast cell lines readily obtained from this biopsy\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Fibroblasts can be reprogrammed to iPSCs by exogenous expression of \u0026ldquo;Yamanaka factors\u0026rdquo; \u003cem\u003eSOX2, cMYC, KLF4\u003c/em\u003e, and \u003cem\u003eOCT3/4\u003c/em\u003e utilizing episomal vectors or non-integrative Sendai virus transduction\u003csup\u003e\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. A major application of this approach is to model genetic disorders as fibroblasts carry the same germline mutation in each patient and after production of iPSCs, can be differentiated to lineages of interest such as neurons, hepatocytes, and cardiomyocytes\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHuman derived primary cells have then greatly expanded the field of biomedical research. In rare disease research, human derived cell models have proven invaluable in understanding disease mechanisms and developing therapeutics\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Recent legislature has also increased interest in the use of iPSCs in drug discovery and clinical trials\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. However, for extremely rare diseases, due to the low number of patients, acquiring such cells has been a challenge for researchers and clinicians. Furthermore, the limiting effects of disease symptoms and the possible financial and medical burdens of travel might prevent participation and contribution of biopsy samples from certain patients and regions. Therefore, any obtained skin biopsy and the resulting fibroblasts are invaluable tools, especially for rare disorders.\u003c/p\u003e \u003cp\u003ePrevious research has demonstrated the applicability to cryopreserve biopsies for later culture\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. However, there is a lack of investigation on the culturing potential of a single biopsy and the effects of extended culture on fibroblast formation and function. We now report a long-term study of biopsy-derived fibroblast cultures including the retained ability to reprogram to iPSCs from long-term fibroblast cultures. Our results indicate that patient-derived biopsies can produce very large numbers of fibroblasts for extended periods of time and these fibroblasts maintain genomic and physiological features. Our results address the ability of long-term cultures to produce patient-derived iPSCs and have broad applications in wound therapeutics and fibroblast physiology.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSkin biopsies\u003c/h2\u003e \u003cp\u003eAll skin biopsies were conducted under the approval of Health Sciences Review Committee #2 of the Institutional Review Board of Vanderbilt University Medical Center, IRB #080369. All research was performed in accordance with relevant guidelines/regulations including those in accordance with the Declaration of Helsinki. Informed consent/assent was obtained from each subject and a single 3 mm punch skin biopsy was obtained from the posterior arm of each subject. This chunk of tissue was dissected in half longitudinally cutting downwards from the exterior surface. Each half was then plated in a single well of a 6 well culture plate. A sterile glass slide cover was placed on the skin chunk. 3 mL of DMEM Complete (DMEM high glutamine, 10% FBS, Pen/Strep) was added to each well, completely submerging the biopsy and slide. Plates were incubated at 37C and 5% CO\u003csub\u003e2\u003c/sub\u003e, gently aspirating and changing media twice a week. After 1\u0026ndash;3 weeks, fibroblast appearing cells began to migrate out. When confluent, skin chunks were moved to a new 6-well plate. Fibroblasts from the original well were then passaged using 0.1% Trypsin-EDTA and plated on a 10 cm petri dish with DMEM Complete for further growth and expansion before cryopreservation.\u003c/p\u003e \u003cp\u003eFor storage, cells were trypsinized and resuspended in fibroblast freezing media (50% DMEM, 40% FBS, 10% DMSO). The cell solution was then aliquoted into multiple cryotubes for each generation, frozen overnight at -80C and then moved to liquid nitrogen for long-term storage. Initial fibroblasts grown from the skin chunk were denoted as the first generation (G1) and subsequent chunk passaging and resulting fibroblast cultures G2-G16.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePatient Selection\u003c/h2\u003e \u003cp\u003eWe focused on a punch biopsy from a male patient with Hikeshi Associated Leukodystrophy (HAL), a devastating neurological disorder caused by homozygous loss of function mutations in the \u003cem\u003eHIKESHI\u003c/em\u003e gene\u003csup\u003e\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Control lines were obtained from first degree relatives with a heterozygous mutation of the \u003cem\u003eHIKESHI\u003c/em\u003e gene. We used male and female first-degree relatives as control lines. The genotype of all lines was confirmed by DNA sequencing and further validated by immunostaining for HIKESHI protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eSkin biopsies were removed from culture plate and fixed in 4% paraformaldehyde (PFA) for a minimum of 24 hours. The biopsies were then transferred into 70% Ethanol and submitted to the Vanderbilt Translational Pathology Shared Resource. Fixed tissue was embedded into paraffin and cut into 5 um thick sections. One set of slides was stained with hematoxylin and eosin (H\u0026amp;E). The H\u0026amp;E-stained slides were submitted to the Vanderbilt Digital Histology Shared Resource and imaged with the Leica SCN400 Slide Scanner (Leica Biosystems) at 40x magnification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eFFPE Slide Immunofluorescence\u003c/h2\u003e \u003cp\u003eTissue sections were deparaffinized and rehydrated by immersion in xylene and a series of ethanol dilutions. Antigen retrieval was performed utilizing heated citrate buffer pH 6. Slides were incubated with blocking buffer for 1 hour at room temperature. Primary antibodies (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were diluted in the blocking buffer and 100 uL added to each well in a humidified chamber at 4C overnight. Secondary antibodies were diluted in blocking buffer and incubated at room temperature for 2 hours. Slides were then washed twice with 1X PBS and mounted using Prolong Antifade DAPI mounting medium (Invitrogen) and allowed to dry overnight before imaging on the EVOS FL Auto microscope.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimary Antibodies used, source, and dilution.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAntibody\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntibody Registry ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUsage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCC3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAB_2341188\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFSP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAB_10000870\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKi67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAB_302459\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:250\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKi67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAB_ 10854564\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:400\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAB_ 2160343\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNanog\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAB_10559205\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOct4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAB_823583\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSSEA3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAB_177628\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSSEA4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAB_528477\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTRA-1-60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAB_2119183\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVimentin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAB_10695459\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eChambered Slide Immunofluorescence\u003c/h2\u003e \u003cp\u003eIbidi 8-well chambered slides (Ibidi 80841) were coated with poly-L-lysine (Sigma Aldrich P4707) for 20 minutes. Poly-L-lysine was aspirated, and slides were washed twice with dH2O and air dried for 2 hours. Fibroblasts were harvested from a confluent T25 flask using 0.25% Trypsin-EDTA solution (Gibco 25200-056) for 7 minutes at 37C before spun down at 300xG for 5 minutes and the pellet was resuspended in 1mL DMEM Complete. A Countess Cell Counter was used cells resuspended in DMEM Complete, plating 2000 cells into each well of the slide. Slides were incubated overnight at 37C. Media was aspirated, and slides were washed with 1X PBS before proceeding with antibody staining as above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell Proliferation Counting\u003c/h2\u003e \u003cp\u003eFibroblasts were plated on 8-well chambered slides at 2,500 cells per well. Cells were fixed and stained for expression of Ki67 and imaged on the EVOS FL Auto microscope at 10x magnification. EVOS Software captured 15 randomized images each from 4 wells. The captured images were analyzed and the total number of DAPI nuclei and Ki67 positive cells were counted. Cell counts were averaged for the well and each well was denoted as a data point for statistical analysis and graphing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eProliferation Assay\u003c/h2\u003e \u003cp\u003eA proliferation assay was conducted on fibroblasts over 7-day period. Fibroblasts were harvested from a confluent T25 flask using 0.25% Trypsin-EDTA solution (Gibco 25200-056) for 7 minutes at 37C. Fibroblasts were spun down, and pellet was resuspended in 1mL DMEM Complete. Cells were counted on the Nexcelom Cellometer Auto T4 Cell Counter and resuspended in DMEM Complete at 10,000 cells/mL. 100 uL of cell solution was added to 8 wells per genotype of 8 96-well plates (1 for each day) for 1,000 cells per well. Plates were incubated at 37C. A Thermofisher CyQuant Direct Cell Proliferation Assay (C35011) was utilized for analysis. For Day 0 to Day 7, the staining solution was prepared per the kit\u0026rsquo;s instructions and 100 uL was added per well. Plates were incubated for 1 hour at 37C. Plates were then read at 485/520nm by the Biotek Synergy HT plate reader. Data and statistics were compiled using Biotek Gen5 and Prism GraphPad.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eRNA Sequencing\u003c/h2\u003e \u003cp\u003eFibroblasts were harvested and resuspended as above. RNA was extracted using the Qiagen RNAeasy Kit (Qiagen 74104) and Qiagen RNA-free DNase kit (Qiagen 79254). Samples were quantified using a NanoDrop and submitted to Vanderbilt Technologies for Advanced Genomics core laboratory.\u003c/p\u003e \u003cp\u003ePaired-end reads were obtained from Illumina NovaSeq6000 PE150 Sequencing. RNA sequence processing was performed with Pyrpipe v0.0.5. First sequence adapters from the reads were trimmed with Trimgalore v0.6.6 and aligned with STAR v2.7.7a to the Genome Reference Consortium Human Build 38 patch release 14 (GRCh38.p14). Assembly of alignments was done with StringTie v2.14 and the GRCh38.p14 NCBI RefSeq annotation assembly (GCF_000001405.40). Gene abundances were estimated using StringTie. Gene and transcript read counts were extracted for each sample with prepDE.py provided by JHU CCB. Pydeseq2 v0.3.5 was used to perform single and multiple factor differential expression analysis among Generations 1 and 6 for both cell lines. Each paired read was treated as a replicate.\u003c/p\u003e \u003cp\u003eGenes with adjusted p-values by the Benjamini and Hochberg method under or equal to 0.001 and log fold change greater than 1 or less than \u0026minus;\u0026thinsp;1 were selected for Gene Ontology Enrichment Analysis (GOEA) using Goatools (v1.3.2)\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. All human genes from Gencode annotations release 39 (GRCh38.p13) served as the background population set. Benjamini-Hochberg p-values were obtained for each Gene Ontology biological process, cellular component, and molecular function pathway. Upregulated and downregulated genes were analyzed separately. Gene associations were taken from the human Gene Ontology 2023 library.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWhole Genome Sequencing\u003c/h2\u003e \u003cp\u003eFibroblasts were harvested and resuspended as above. DNA was extracted using the DNAeasy Kit (Qiagen 69504). Samples were quantified using a NanoDrop. Samples were submitted to CD Genomics for low-pass whole genome sequencing and analysis. Samples were run through a quality test then utilized to create a construct library. The generated library was sequenced using the Illumina HiSeq sequencing platform producing raw data. The raw data was processed to filter out various reads including low quality and redundant reads. The sequencing quality score is determined, and the distribution of base content is calculated from the raw data. The reads were then aligned with a reference genome and Copy Number Variations (CNV) determined. Deletions and duplications were identified and annotated with gene names or Ensembl IDs. Chromosome diagrams were generated for each genotype depicting the CNVs. Statistics were conducted throughout the process for sequencing reads, alignment results and more.\u003c/p\u003e \u003cp\u003eGOEA was performed in the same manner as with gene expression using DNA duplications and deletions from Generation 16 fibroblasts. Both duplications and deletions were considered a change to the gene and used as the study population. Changes already present in Generation 1 were removed from this set. A p-value of 0.001 or less was used to identify modified pathways.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFibroblast Reprogramming\u003c/h2\u003e \u003cp\u003eFibroblasts were reprogrammed using the CytoTune-iPS 2.0 Reprogramming Kit (Invitrogen A16517). Fibroblasts were plated at 2x10\u003csup\u003e5\u003c/sup\u003e cells per well of a Matrigel-coated 6-well plate. Cells were fed with DMEM Complete. The following day (Day 0), 1 well was disassociated from the plate and the cell number was counted. The volume of virus required was calculated using the formula:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$Vol of virus \\left(ul\\right)=\\frac{MOI x cells/ml}{\\begin{array}{c}\\frac{CIU}{ml}x\\frac{{10}^{-3}ml}{ul}\\\\ \\end{array}}$$\u003c/div\u003e\u003c/div\u003e.\u003c/p\u003e \u003cp\u003eViruses hKOS and cMYC had a MOI of 5 and KLF4 a MOI of 3. Virus was added to DMEM complete w/o PenStrep, adding 1 mL per well of cells. The following day (D1), the virus solution was carefully removed, and the cells were fed with DMEM Complete, changing the media every day from Day 1 to Day 6.\u003c/p\u003e \u003cp\u003eOn Day 7, the cells were disassociated with 0.25% Trypsin and counted. They were plated on Matrigel-coated 6 well plates with densities of 5x10\u003csup\u003e4\u003c/sup\u003e, 7.5X10\u003csup\u003e4\u003c/sup\u003e and 1x10\u003csup\u003e5\u003c/sup\u003e cells per well. The cells were moved to Essential 8 Medium (Gibco A15169-01) with Supplement (Gibco A15171-01). Cells were then grown in E8 Medium, feeding every other day from Day 7 to Day 21. Cells were then moved to mTesR1 media (StemCell Technologies #85857). Individual colonies were identified and picked by scraping, colonies were transferred to a Matrigel-coated 48 well plate and grown on mTeSR1. Colonies were passaged over multiple generations and then fixed with 4% PFA for 30 minutes at room temperature. Immunofluorescence on colonies was done as above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eRNA Sequencing data was deposited in Gene Expression Omnibus (GEO) under accession GSE271347.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eTo generate primary dermal fibroblast cultures, for years we have been obtaining skin biopsies from patients with rare neurological disorders. The fibroblasts are used for experiments and for reprogramming to iPSCs. Given the very rare nature of some disorders we have studied, the initial skin biopsy from a patient with Hikeshi Associated Leukodystrophy (HAL, OMIM # 616881) was propagated further after initial fibroblasts were collected. We had previously observed the potential of human skin chunks to continue to generate fibroblasts when the biopsy was placed in a new well. As patients with HAL can decompensate and even die following fever and infection, we strove to limit exposure to any invasive procedures including skin biopsy. We continued to propagate this individual chunk of skin for greater than 15 months. We noted the continued generation of fibroblasts, even from latter generations. We hypothesized that long-term culture of the skin chunk did not impede fibroblast identity and function including cell morphology, gene expression, and ability to be reprogrammed to iPSCs. The HAL patient biopsy (HIKHom-1) was propagated for 16 generations with many tubes of fibroblasts frozen down at each passage upon transfer of the skin chunk to a new well. Control cultures derived from first-degree relatives with heterozygous \u003cem\u003eHIKESHI\u003c/em\u003e mutations (HIKHet-2) were also propagated, summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSkin Biopsies used and Fibroblast Generations Post Biopsy.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLabel\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGeneration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDays since biopsy\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHetG1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHIKHet-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9 days\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHetG6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHIKHet-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e159 days\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHomG1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHIKHOM-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e71 days\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHomG6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHIKHOM-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e190 days\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHomG16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHIKHOM-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e473 days\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAfter final generation of fibroblasts, the HIKHOM-1 and HIKHet-2 skin chunks were harvested, fixed, and sectioned. H\u0026amp;E stains of HIKHOM-1 and HIKHet-2 were compared to two unrelated control skin biopsies harvested after one generation. Likely due to age (G16 versus G6) the HIKHOM-1 skin biopsy appeared thinner and more damaged compared to control skin biopsies. The HIKHet-2 biopsy was also thinner and appeared more damaged than the controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAll fibroblasts regardless of age, generation, and genotype exhibited the characteristic spindle morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), swirled patterns of growth, and contact inhibition when cultures were confluent\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Therefore, the cells seemed to retain fibroblast lineage and morphology over various generations, suggesting that long-term biopsy culture does not affect the cell type or morphology.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs we hypothesized that resident progenitor cells within each piece of skin remained proliferative and capable of producing fibroblasts, we used immunofluorescence for expression of Fibroblast-Specific Protein 1 (FSP1), a member of the S100 calcium-binding protein superfamily\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. FSP1 is present in fibroblasts and absent in epithelial, mesangial, and embryonic endoderm cells\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Cells were also stained for the cytoskeletal protein Vimentin, which has also been frequently used as a fibroblast marker\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The staining displayed that the cells had both the spindle-shaped body associated with inactive fibroblasts and the larger, more stellate bodies of active fibroblasts.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCell Proliferation and Death\u003c/h2\u003e \u003cp\u003eWe thawed fibroblasts from different generations and genotypes and measured proliferation over 7 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). There were no significant changes in proliferation between HIKHet-2 G1 and HIKHet-2 G6 over 7 days. However, there was a significant decrease in proliferation between HIKHOM-1 G1 and HIKHOM-1 G6 Day 6 onwards (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Furthermore, there was a significant decrease in proliferation in HIKHOM-1 G16 compared to both HIKHOM-1 G1 and HIKHOM-1 G6 from Day 4 onwards. It was also observed that the HIKHOM-1 cell lines overall had a higher proliferative rate than HIKHet-2 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine the percentage of proliferating cells, cells were fixed and stained for a proliferation marker, Ki67\u003csup\u003e42\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Cell counts indicated that the percentage of Ki67 expressing cells decreased over generations. There was a downward trend between Gen1 and Gen6 of both HIKHet-2 and HIKHOM-1 genotypes, however this decrease was not significant. There was a significant decrease between HIKHOM-1 G1 and HIKHOM-1 G16, suggesting that long-term culture slows but does not halt proliferation by 473 days. Similar to the proliferative assay, the HOM genotype displayed a higher percentage of Ki67 positive cells across all generations when compared to the Het cells.\u003c/p\u003e \u003cp\u003eTo analyze the presence of proliferative cells in the biopsy samples after long-term culture, the biopsies were ultimately fixed, paraffin-embedded, and immunostained for proliferative markers. Immunofluorescence staining indicates a lack of Ki67 positive and PCNA positive cells in HIKHOM-1, HIKHet-2, and unrelated control skin biopsies (data not shown). Validity of this assay was confirmed utilizing multiple antibodies and positive control tissue. Cells were also fixed and stained for the apoptosis marker, Cleaved Caspase 3 (CC3)\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Fibroblasts were negative for CC3 indicating minimal if any apoptotic cell death (data not shown). Antibody validity was confirmed utilizing positive control cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eGene Expression and Genomic Integrity\u003c/h2\u003e \u003cp\u003eTo determine if there were appreciable differences in gene expression, RNA was obtained from proliferating fibroblast lines from different generations and genotypes. RNA sequencing was conducted, and we observed significant upregulation and downregulation of various genes between HIKHOM-1 G1 and G16 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Various genes such as \u003cem\u003eHAPLN1, LHX\u003c/em\u003e, and \u003cem\u003eSDK2\u003c/em\u003e were highly upregulated and \u003cem\u003eSMO, C3\u003c/em\u003e, and \u003cem\u003eEMILIN2\u003c/em\u003e were highly downregulated. Gene enrichment analysis between HIKHOM- 1 G1 and G16 indicated that a variety of pathways were changed. Pathways relating to the extracellular matrix, ion channel activity, and transmembrane transport were upregulated. Pathways relating to the regulation of the immune system, response to stimulus, and cell surface receptor signaling were downregulated. Analysis between HIKHOM-1 G1 and G6 as well as HIKHet-2 G1 and G6 also displayed changes in expression and gene enrichment, but substantially fewer changes to expression and pathways were observed implying long-term culture amplified transcriptional changes (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs prolonged time in culture could predispose cells to genomic instability and acquisition of mutations, we processed DNA from proliferating fibroblast lines from different generations and genotypes for low-pass whole genome sequencing. This allowed us to assess chromosomal stability of long-term culture, and to detect any large DNA sequence gains or losses. Chromosomal maps indicate that there were no significant chromosomal gains or losses that developed over generations (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eFurther enrichment analysis determined that between HIKHom1 G1 and G16, only a single pathway was significantly changed. A deletion in Chromosome 22 affected genes in the \u003cem\u003eAPOBEC3\u003c/em\u003e cluster including \u003cem\u003eAPOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D\u003c/em\u003e and \u003cem\u003eAPOBEC3F\u003c/em\u003e. This resulted in potential reductions in deaminase activity and negative regulation of viral processes\u003csup\u003e\u003cspan additionalcitationids=\"CR45 CR46\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Despite these genomic changes, the cells displayed no increased susceptibility to contamination. The genomic analysis also indicated that mutations occurred at random and no specific pathway, such as cancer-related pathways were targeted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eReprogramming Competence\u003c/h2\u003e \u003cp\u003eTo determine if prolonged propagation of skin biopsies could produce fibroblasts that are competent for reprogramming to iPSC, HIKHet-2 G1 and HIKHOM-1 G16 fibroblasts were reprogrammed using Sendai virus containing Yamanaka factors. By Day 16, colonies of round shaped cells were evident as well as surrounding cells maintaining a typical fibroblast morphology. On Day 21 since transduction with Sendai viruses, emerging iPSC-like colonies were manually picked and replated. These were grown out for a few more days before being imaged. The individual colonies were flat and consistent with iPSC morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), consisting of small, round cells with high nuclear/cytoplasmic ration with prominent nucleoli\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo provide further evidence that HIKHOM-1 G16 fibroblasts were competent for reprogramming, individual colonies were immunostained for pluripotency markers Nanog, Oct4, TRA-1-60, SSEA3, and SSEA4\u003csup\u003e49,50\u003c/sup\u003e. All lines stained positive for all markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), indicating pluripotent status and successful reprogramming.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study demonstrates the long-term viability and reprogramming competency of skin biopsy derived fibroblast cultures. A prolonged 16-month culture of a single 3 mm skin biopsy produced 16 generations of fibroblasts amounting to potentially billions of cells. These fibroblasts have been utilized for a variety of experiments including immunofluorescence, immunoblotting, RNA and DNA sequencing, and iPSC reprogramming. We observed retention of fibroblast morphology and marker expression, further supporting the long-term maintenance of the cellular identity.\u003c/p\u003e \u003cp\u003eProliferation assays showed that later generations have decreased proliferative capacity compared to the first generation of fibroblasts. HIKHOM1 fibroblasts did show an overall higher level of proliferation compared to HIKHet2 cells which could be due to genotype as well as age differences \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. At the end of the prolonged culture period, we did not observe proliferative cells in the fixed and sectioned skin biopsies. Due to the small number of cells visible in the biopsy, this could be due to the tissue sections lacking proliferative cells. Previous studies have also shown that extended formalin-fixation can reduce the Ki67 staining\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Cultured fibroblasts were negative for cleaved caspase 3 (CC3) cells indicating minimal if any apoptotic cell death. As plated cells were not confluent, the fibroblasts appear to be continuously proliferative but exhibited contact inhibition\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. As the cultured fibroblasts were passaged or frozen upon confluency, this could result in a lack of apoptotic pressure on the cells.\u003c/p\u003e \u003cp\u003eRNA and DNA sequencing demonstrate the overall maintenance of genomic integrity over long-term culture. There were no chromosomal abnormalities over fibroblast generations. The few acquired mutations seem to have occurred at random and did not appear to specifically target a pathway or affect the physiology and culture potential of the cells. Studies on other cell types such as mesenchymal stem cells have demonstrated that long-term culture can result in random mutation accumulation with oxidative stress of culture conditions playing a primary role\u003csup\u003e\u003cspan additionalcitationids=\"CR56\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Further experiments would determine if regulation of culture conditions to reduce oxidative stress will reduce mutation frequency in long-term skin-biopsy culture. Extensive changes in expression were observed between the first and last generation, similar to studies observing long-term culture of mesenchymal stem cells\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. The resulting protein changes and functional differences caused by these expression changes will require further study. Successful reprogramming of G16 fibroblasts indicates that reprogramming competence was not altered over long-term culture of fibroblasts. Due to the DNA and RNA changes, reprogrammed stem cells would have to be further sequenced and propagated to determine that the apparent randomly acquired changes do not affect stem cell physiology and differentiation potential. If long-term fibroblast culture is utilized in clinical applications, both directly and for reprogramming, cells will have to be frequently sequenced to ensure that no harmful mutations have been acquired that may reduce therapeutic potential or cause a host response.\u003c/p\u003e \u003cp\u003eIn this report we determined the prolonged capacity and robustness of skin biopsies to continuously produce many generations of primary fibroblasts. Especially for rare genetic diseases, these findings have important implications in primary cell culture and \u003cem\u003ein vitro\u003c/em\u003e translational research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding Sources:\u003c/h2\u003e \u003cp\u003eInstitutional Funding from Vanderbilt University Medical Center and University of Colorado Anschutz Medical Campus\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConflict of Interest:\u003c/strong\u003e \u003cp\u003eThe authors declare no conflicts of interest\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSRK and KCE conceived and designed the study. SRK, KCE, and GMW performed experiments as well as data collection. SRK, MAR, and KCE analyzed the data and interpreted the results. SRK and KCE wrote the manuscript. All authors reviewed the results and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank the patient with HAL and their family for allowing us to obtain skin biopsies. We also thank our funding sources who wish to remain anonymous. We thank Brittany Parker Short and Tenhir Iyer for technical assistance. Whole slide imaging was performed in the Digital Histology Shared Resource at Vanderbilt University Medical Center (www.mc.vanderbilt.edu/dhsr). Slide production was performed by Translational Pathology Shared Resource, supported by NCI/NIH Cancer Center Support Grant P30CA068485.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eRNA Sequencing data was deposited in Gene Expression Omnibus (GEO) under accension GSE271347.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePlikus M V., Wang X, Sinha S, et al. 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Molecular Therapy Nucleic Acids. 2021;26:557\u0026ndash;574. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.omtn.2021.08.013\u003c/span\u003e\u003cspan address=\"10.1016/j.omtn.2021.08.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Fibroblasts, reprogramming, skin culture","lastPublishedDoi":"10.21203/rs.3.rs-4651236/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4651236/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePrimary fibroblasts are a commonly used cell type used in a variety of experimental and therapeutic studies. Patient-derived skin biopsies are an accessible way to generate fibroblasts for use in various assays as well as reprogramming to iPSCs. To understand the potential of long-term skin biopsy culture, we cultured biopsy samples for 6 to 16 months and analyzed subsequent generation of fibroblasts. We found maintenance of morphology and physiology over time. Proliferation assays showed that older generations remained proliferative but at a decreased rate. mRNA analyses revealed transcriptional changes with long-term skin culture. Deep DNA sequencing did not reveal any large deletions or amplifications. Spontaneous DNA mutations seemed to be random and not enriched for any specific signaling pathways. Older fibroblasts generated after 16 months in culture retained competency for reprogramming into iPSCs. Our results support long term culture of skin to generate very large numbers of primary fibroblasts. These cells maintain their identity and integrity enabling the study of human disorders, particularly rare ones.\u003c/p\u003e","manuscriptTitle":"Long-term culture of skin biopsies: Maintenance of fibroblast production and competency of reprogramming","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-26 21:08:52","doi":"10.21203/rs.3.rs-4651236/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9085f144-8e97-4e7d-8f07-647d2ae48d72","owner":[],"postedDate":"July 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":34839667,"name":"Biological sciences/Stem cells/Mesenchymal stem cells"},{"id":34839668,"name":"Biological sciences/Biological techniques/Cytological techniques/Tissue culture"}],"tags":[],"updatedAt":"2026-02-17T19:21:25+00:00","versionOfRecord":{"articleIdentity":"rs-4651236","link":"https://doi.org/10.1002/2211-5463.70136","journal":{"identity":"febs-open-bio","isVorOnly":true,"title":"FEBS Open Bio"},"publishedOn":"2025-12-19 00:00:00","publishedOnDateReadable":"December 19th, 2025"},"versionCreatedAt":"2024-07-26 21:08:52","video":"","vorDoi":"10.1002/2211-5463.70136","vorDoiUrl":"https://doi.org/10.1002/2211-5463.70136","workflowStages":[]},"version":"v1","identity":"rs-4651236","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4651236","identity":"rs-4651236","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}