Ruth J. Mathew 1, Yongchao Mou, Ph.D.2, Zhenyu Chen, Ph.D.2 Xue-Jun Li, PhD2
1 University of Illinois, Chicago, IL
2 Department of Biomedical Sciences, University of Illinois College of Medicine at Rockford, Rockford, IL
Abstract
Hereditary Spastic Paraplegia (HSP) is a group of inherited neurodegenerative disorders caused by axonal degeneration of the cortical motor neurons. HSP affects around two out of 100,000 people worldwide. Symptoms of HSP involve spasticity, numbness and tingling sensations in the lower limb muscles. The most common recessive form of HSP, Spastic Paraplegia Type 15 (SPG15), represents 2-4% of HSP cases. SPG15 is correlated with mutations in the ZFYVE26 gene, potentially leading to lysosomal dysfunction in cortical motor neurons, resulting in mitochondrial impairment and consequent axonal degeneration. However, the challenges of acquiring patient neurons require the application of induced pluripotent stem cells (iPSCs). iPSCs provide an innovative approach to studying patient-specific mutations and disease phenotypes. This study aims to determine the pluripotency of SPG15 iPSCs derived from patient-specific fibroblasts, serving as the first crucial step in deriving stem cell models that can later be used to test for inhibitors of axonal degeneration. Following the generation of SPG15 iPSCs from patient-specific fibroblasts, we tested both samples for multiple genes and protein expressions using regular PCR, qPCR and immunostaining. The results of [which test?] indicate that the iPSC sample had elevated levels of the pluripotent genes NANOG, OCT4 and SOX2, as well as pluripotent proteins, OCT4, SSEA4, NANOG and SOX2 in comparison to the initial patient fibroblast samples. In parallel, the expression of fibroblast gene FGF5 in the iPSCs was reduced to about 0.21% of its initial expression in fibroblast cells from the Student’s t-test. The confirmation of cell pluripotency makes the SPG15 iPSCs suitable for future studies to characterize differentiated SPG15 cortical motor neurons and analyze the effects of HSP SPG15 treatments on neural cells.
Introduction
Hereditary Spastic Paraplegia (HSP) is a collective group of neurodegenerative diseases that involves several impairments, encompassing abnormal organelle morphology and regeneration, imbalanced lipid and steroid levels, lysosomal degeneration and mitochondrial dysfunction in neural cells (Blackstone et al. 2011). Previous research investigating diseased HSP neurons has highlighted an imbalance of mitochondrial fission and fusion processes that can inhibit mitochondrial mobility along neurons (Denton 2018). The imbalance results in various axonal defects such as reduced axonal outgrowth, decreased mitochondrial density, increased axonal swelling, and cellular apoptosis (Denton 2018).
Spastic Paraplegia Type 15 (SPG15) is the second most common recessive variant of HSP, representing 2-4% of HSP cases and involving symptoms such as numbness, tingling, and pain in lower limb muscles (Denton et al. 2016; U.S. National Library of Medicine 2021). The symptoms of SPG15 are connected to mitochondrial dysfunction in cortical motor neurons. As an inherited disorder, SPG15 is caused by mutations in the ZFYVE26 gene that encodes for SPASTIZIN; This protein aids in lysosomal functions, axonal transport and cytokinesis in neural cells (Ebrahimi-Fakhari et al. 2021; Blackstone et al. 2011). With inhibited SPASTIZIN lysosomal functions, neural cells can undergo impaired autophagy processes, resulting in accumulations of protein aggregates, thereby leading to various symptoms such as axonal degeneration (Vantaggiato et al. 2023). Current HSP SPG15 treatments consisting of various physical exercises and oral antispasmodics, such as baclofen and tizanidine, have not been proven to significantly inhibit HSP symptoms (Shribman et al. 2019).
Studies relying on diseased post-mortem neurons often face challenges, as these neurons are difficult to obtain, maintain and utilize in characterizing the early-stage development of neurodegenerative disorders. Thus, the use of induced pluripotent stem cells (iPSCs) provides an avenue for observing the gradual progression and phenotypic characterization of neurodegenerative diseases (Karpe et al. 2021). Additionally, high self-renewal and differentiation capacity allow iPSCs to differentiate into cortical disease projection neurons, which have been useful in studying mitochondrial irregularities that can influence neural degeneration (Chamberlain et al. 2008).
Despite being generated for some forms of HSP, a pluripotent patient-derived stem cell model has not yet been fully utilized to study the SPG15 variant of HSP (Denton 2018). Therefore, this study aims to test for the pluripotency of SPG15 iPSCs that have been derived from patient-specific fibroblasts using Sendai viruses, a safer method for introducing the transduction factors to the fibroblast cells that minimizes risks for modifying or mutating the host genome. In comparison to retroviruses and lentiviruses, Sendai viruses replicate as single-stranded RNA in the cytoplasm without producing or integrating DNA into the cell genome. Therefore, stem cell reprogramming through Sendai viruses effectively transduces the necessary pluripotent transcription factors without the integration of genetic mutations that might affect cellular function (Kunitomi et al. 2022). Testing for cell pluripotency is the first crucial step in deriving stem cell models that can later be differentiated into cortical projection neurons to test for inhibitors of mitochondrial dysfunction and axonal degeneration in patients affected by SPG15. The genetic expression of pluripotent transcription factors, such as OCT4, SOX2 and NANOG, as well as pluripotent proteins such as SSEA4 and TRA-1-60 can be analyzed to validate the generated stem-cell pluripotency model (Takahashi et al. 2007). Additionally, a minimized expression of fibroblast growth factors (FGF) in the iPSCs can verify the reprogramming of the fibroblasts into pluripotent stem cells. If the SPG15 iPSCs express certain genes and proteins that indicate cell pluripotency, then the model can be validated as pluripotent, or having the ability to differentiate into different cell lines. These include neural cells that can be utilized in testing for potential inhibitors of axonal degeneration.
Materials and Methods
Deriving iPSC from Fibroblasts Using Sendai Viruses
To derive iPSC colonies from patient-specific fibroblasts, we used Sendai virus-mediated reprogramming. The fibroblast cells were infected with Sendai viruses and cultured in a fibroblast cell medium (2mL medium per well, changed every other day). After one week of culture, the fibroblasts were passaged onto Mouse Embryonic Fibroblasts (MEFs) and cultured in a 6-well plate (at a density of about 0.1 million cells per well) in Human Embryonic Stem Cell (hESC) medium. Once patient-specific fibroblasts were collected, SPG15 iPSCs were generated by transducing the cells via Sendai viruses that contain the four reprogramming factors (OCT3/4, SOX2, KIF4, L-myc). After the fibroblasts were infected with the Sendai viruses and cultured in a fibroblast culture medium, some fibroblasts were passaged onto MEFs and cultured in hESC medium. After approximately twenty-four days, iPSC colonies began to form, suspended in solution. These colonies were to be isolated, counted and used for gene/protein characterization and further neural differentiation. For this study, SPG15 iPSC RNA was isolated and then converted to cDNA using the enzyme reverse transcriptase in order to characterize the SPG15 iPSCs based on pluripotent gene expression.
Genetic Testing
After the generated SPG15 stem cells’ RNA was isolated, the cells were tested for stem cell gene expression by utilizing standard PCR and Gel Electrophoresis (Thermo Fisher Scientific, Waltham, MA). The Fibroblasts and SPG 15 iPSCs cDNA samples were mixed with 6µL of Go Taq solution and 1µL of primers to make a 12µL PCR mixture, which was then run for 32 cycles at an annealing temperature of 60°C before loading the samples into a 1.5% agarose gel for gel electrophoresis (90 Volts for 30 minutes).
The samples were tested for the housekeeping gene, GAPDH, to establish the appropriate concentration of cDNA needed to test for the expression of pluripotent transcription factors– NANOG, OCT4 and SOX2– and the gene FGF5 (Fibroblast Growth Factor 5), compared by the relative brightness of the samples under a gel imager. Biological replicates were utilized to validate gene expression levels, a representative replicate is included in Figure(s) 1a-c. After using standard PCR to test for the mRNA expression of transcription factors, qPCR (n = 3) was used to confirm the gene expression of FGF5 in both Fibroblast and SPG15 iPS cDNA samples using the CT values data and the calculated fold difference values. The statistical differences between the fibroblast and SPG15 cell groups were examined using a two-sided t-test. The significance level was defined as p < .05.
Protein Analysis
Immunostaining was used to analyze the protein expression of pluripotent proteins such as OCT4, SSEA4, NANOG and SOX2 in the SPG15 iPSCs. The cell samples were fixed, stained using primarily antibodies against these proteins (e.g., anti-OCT4, mouse-IgG from Santa Cruz Biotechnology) and fluorescence-conjugated secondary antibodies (anti-mouse-IgG), then imaged for analysis.
Results
After establishing an equal expression of the housekeeping gene, GAPDH, in the fibroblast and SPG15 iPSC sample (Figure 1a), it was evident that the SPG15 iPSCs had a high expression of the pluripotent transcription factors—OCT4, SOX2 and NANOG—compared to the fibroblasts, which had almost no expression of the pluripotent genes. Gene expression was compared using replicated samples of fibroblast and SPG15 iPSCs. The pluripotent transcription factor expression observed through gel electrophoresis demonstrated the iPSCs have a high expression of the genes OCT4, NANOG and SOX2, each approximately 100-200bp. The high expression of genes helped confirm the pluripotency of the reprogrammed stem cells (Figure 1c). In comparison, the fibroblasts expressed the gene FGF5 significantly greater than the iPSCs, further suggesting that the fibroblasts have been fully reprogrammed with the transduction of the four reprogramming factors (Figure 1b). The FGF5 gene expression was quantified using qPCR. Statistical analysis of qPCR results using a two-sided t-test (p < .01) indicates that the SPG15 iPSC sample had a significant reduction in the expression level of 0.0021 (0.0021 + 0.0019) as compared to the fibroblast cell sample (expression level of 1 + 0.112). Thus, the fibroblast cells had a much higher expression of the fibroblast gene, FGF5, when compared to the generated iPSCs, validating the regular PCR results (Figure 1d). The results from the PCR and qPCR tests, including the expression of pluripotent genes for the iPSCs and fold difference in the expression of the gene FGF5, collectively confirm the reprogramming of the SPG15 fibroblasts into pluripotent stem cells.
Figure 1. a. Gene expression in fibroblast and SPG15 iPS cell samples. In setting a baseline sample concentration, there is an equal expression of the housekeeping gene, GAPDH, in both the fibroblast and SPG15 iPS cell samples as established using gel electrophoresis, signifying sufficient cDNA concentrations have been attained to continue with testing genetic expression. b. Gel electrophoresis results. The results from gel electrophoresis indicate a greater relative gene expression of the gene FGF5, a marker for fibroblast cells for loss-of-origin validation, in the fibroblast cells as opposed to the SPG15 iPSC cDNA samples. c. Highlighted gene expression. Similarly, there is a greater relative gene expression of the pluripotent transcription factors — OCT4, NANOG, and SOX2 — in the SPG15 iPSC samples as opposed to the fibroblasts. d. FGF5 fold difference and standard deviation. The fold difference and standard deviation calculated from the quantitative PCR results confirm the pluripotency of the SPG15 iPSCs. SPG15 iPSC: 0.0021±0.0019, **p < 0.01 versus Fibroblasts.
Discussion
With the purpose of creating a pluripotent stem model for analyzing diseased SPG15 neurons, the performed genetic expression analysis confirms the validity of SPG15 fibroblast-derived iPSCs through the clear expression of genes and proteins demonstrating cell pluripotency. The highly increased expression of pluripotent genes (OCT4, NANOG and SOX2) and proteins (OCT4, SSEA4, NANOG and SOX2) in comparison to the original fibroblast cells validates the potency and identity of the SPG15 iPSCs, making them suitable for future studies to characterize differentiated SPG15 cortical motor neurons. For HSP treatment development, the patient-specific iPSCs can be differentiated into cortical motor neurons and tested with possible therapeutic agents against axonal degeneration as seen in the study of other variants of HSP, including SPG11 (Chai et al. 2023). Additionally, one can use mitochondrial tracking and lysosomal flux assays in differentiated cortical motor neurons to characterize the dynamics and role of these processes in neurodegeneration.
The pluripotent stem cells serve as a disease model that can be valuable for future studies determining the genetic and cellular alterations resulting from any plausible axonal degeneration treatment. Some limitations of using a stem-cell derived model include the challenges of achieving full maturation in vitro, replicating in vivo neural conditions and addressing variability between cell populations. However, the difficulty of acquiring patient neurons and the variability between patient lines limits reproducibility. Stem cell models provide an innovative approach to studying neurodegeneration that bypasses the challenges in reproducibility. Thus, differentiated cortical cells can be utilized to further explore the causes and characteristics of SPG15 and other neurodegenerative diseases through in vivo studies in which the effects of the diseased cells can be observed in mice.
Gaining a deeper understanding of the disease itself using the patient-specific iPSCs is crucial to developing a treatment that effectively targets mitochondrial dysfunction that leads to axonal degeneration in cortical motor neurons (Mou et al. 2019). In addition to addressing neurodegenerative diseases, the method of iPSC generation from patient-specific fibroblasts through Sendai viruses presents a new and safer method of stem cell reprogramming without the integration of potential exogenous transgenes into the cell genome (Kunitomi et al. 2022). This study further confirms the effectiveness of Sendai viruses in deriving stem cell models for SPG15, expressing pluripotent genes and proteins that can be used to study disease-specific mutations and axonal degeneration. In the future, we hope to differentiate these stem cells into cortical neurons and study disease phenotypes, mitochondrial transport and expression of neuronal markers.
Conclusions
This study focused on confirming the pluripotency of differentiated HSP SPG15 iPSCs. Through our study, we were able to generate pluripotent SPG15 iPSCs from patient-specific fibroblast cells by using Sendai viruses containing reprogramming factors. Gene and protein testing confirmed the pluripotency of the generated stem-cell model through the presence of pluripotent genes and proteins. In the future, these cells can be differentiated into SPG15 cortical neurons to be characterized and experimented with different HSP treatments.
Acknowledgment
The authors acknowledge the 2021 Summer Science Internship at the University of Illinois College of Medicine at Rockford and the Blazer Foundation.
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