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 confluent41. 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 superfamily37. FSP1 is present in fibroblasts and absent in epithelial, mesangial, and embryonic endoderm cells38. Cells were also stained for the cytoskeletal protein Vimentin, which has also been frequently used as a fibroblast marker39,40 (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. 3A). 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. 3A). 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, Ki6742 (Fig. 3B). 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 processes44–47. 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. 5A), consisting of small, round cells with high nuclear/cytoplasmic ration with prominent nucleoli48.
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 SSEA449,50. All lines stained positive for all markers (Fig. 5B), indicating pluripotent status and successful reprogramming.