Applying hydrodynamic pressure to efficiently generate induced pluripotent stem cells via reprogramming of centenarian skin fibroblasts
Abstract
Induced pluripotent stem cell (iPSC)-technology is an important platform in medicine and disease modeling. Physiological degeneration and disease onset are common occurrences in the aging population. iPSCs could offer regenerative medical options for age-related degeneration and disease in the elderly. However, reprogramming somatic cells from the elderly is inefficient when successful at all. Perhaps due to their low rates of replication in culture, traditional transduction and reprogramming approaches with centenarian fibroblasts met with little success. A simple and reproducible reprogramming process is reported here which enhances interactions of the cells with the viral vectors that leads to improved iPSC generation. The improved methods efficiently generates fully reprogrammed iPSC lines from 105–107 years old subjects in feeder-free conditions using an episomal, Sendai-Virus (SeV) reprogramming vector expressing four reprogramming factors.
In conclusion, dermal fibroblasts from human subjects older than 100 years can be efficiently and reproducibly reprogrammed to fully pluripotent cells with minor modifications to the standard reprogramming procedures. Efficient generation of iPSCs from the elderly may provide a source of cells for the regeneration of tissues and organs with autologous cells as well as cellular models for the study of aging, longevity and age-related diseases.
Introduction
Aging is accompanied by a significant decline in physiologic functions in several organs, and by a dramatic increase in disabilities. At the cellular level, a part of this decline is related to cell senescence [1,2]. During the past years, the scientific community faced an increasing demand in cell-based technologies aimed at treating disorders associated with aging to enable elderly people to lead healthy and more productive lives [3]. The introduction of cell fate-manipulating technologies for the remodeling of somatic cells into embryonic-like stem cells has opened the door to new studies in geriatric disorders. Human induced Pluripotent Stem Cells (iPSCs) have the potential to provide a nearly unlimited source of cells for basic research, and disease modeling [4].
IPSCs have been generated from a multitude of somatic cell types deriving either from fetal, pediatric or adult tissues [5]. In general, cell reprogramming is achieved by over-expressing specific embryonic-state regulating transcription factors (i.e. OCT4, SOX2, KLF4, NANOG) through transduction of exogenous copies of the overmentioned genes. Different transduction methods have been used to generate iPSCs, including viral vectors (retro-, adeno-, lenti- and sendai-virus), bacterial artificial chromosomes (BAC) system, episomal vector transfection and mRNA and protein-based delivery systems (for review see [6,7]). Retrovirus- or lentivirus-mediated gene delivery methods have been employed although integration of the exogenous vector into the host genome could lead to mutagenesis [8]. Recently, a viral approach using non-integrating sendai virus (SeV) has been proposed [9]. In SeV reprogramming, transgenes remain episomal and are lost as cell proliferate. Compared to the other methods, SeV reprogramming resulted in efficient generation of hiPSCs with fewer genetic abnormalities and genotoxicity [10,11].
The age of the donor from which the somatic cells were derived influences the efficiency of iPSC reprogramming [12–14]. Fibroblasts from young mice with a high proliferation rate were reprogrammed more efficiently than were cells from older animals. In addition, iPSCs derived from old mice lost pluripotency features during serial passages [15]. Cellular senescence increases with age and is often described as being associated to an irreversible arrest in cell cycle, induced by p53/p21 and p16 activation [1,16,17]. Expression of p16 and p21 is up-r+egulated in cells from most elderly donors, resulting in reduced proliferation. The overexpression of p16 and p21 increases the chance of initiation of internal senescence programs and limits the capacity of cells to be reprogrammed [18]. The suppression of p53/p21 pathway by specific siRNA/shRNA, was shown to increase the efficiency in iPSC generation [19,20]. To overcome senescense pathways, directed overexpression of NANOG and LIN28 in combination with standard Yamanaka factors (OCT3/4, SOX2, KLF4, c-MYC) has been proposed to facilitate hiPSC generation. Thus, a six-factor protocol has been reported to be necessary to generate human iPSC from centenarian fibroblasts [21,22].
The aim of our study was to optimize and simplify a protocol to efficiently reprogram somatic cells isolated from aged donors with a minimum number of reprogramming factors, without genome integration and under feeder-free conditions.
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Discussion
Cellular reprogramming is a gradual course in which expression of endogenous genes linked to pluripotency are induced in somatic cells. However, such an approach is often inefficient, with limited success and generation of hiPSC was reported to be a stochastic process, where most of the differentiated cells fail to acquire a pluripotent state. [25–27].
Here we report a reliable method to generate fully reprogrammed iPSC lines from human dermal fibroblasts isolated from exceedingly old individuals. Even with the improved methods shown here, the efficiency of reprogramming somatic cells derived from centenarians remains significantly lower than younger cells as previously reported [18]. However, when successful, generated cells met the criteria of fully reprogrammed cells: 1) they present a stable hES-like colony morphology over several passages; 2)show Alkaline Phosphatase activity; 3) express pluripotency markers OCT4, SOX2, NANOG, TRA 1–60, TRA1 1–81 and SSEA4 (confirmed either at the gene and/or protein level); 4) present a pluripotent transcriptomic profile (as shown by PluriTest comparison); 5) lack large chromosomal abnormalities. Presumably an irreversible cell cycle arrest leads to a lower efficiency in cellular reprograming [16,19]. Procedures involving p53 pathway inhibition by specific siRNA/shRNA have resulted in efficient reprograming of somatic cells derived from centenarian donors [28]. Another strategy, based on genetic manipulation by forced expression of 6 reprogramming factors including NANOG and LIN28, increased cellular division and reprogramming capacity [21]. These data suggested that rather extraordinary measures and additional epigenetic manipulation in addition to the standard Yamanaka’s factors may be necessary to efficiently reprogram centenarian derived cells. Reducing the volume of medium used during the viral exposure and applying hydrodynamic pressure by centrifugation of the culture plates facilitate attachment and integration of viral particles allowing the generation fully reprogrammed cells from centenarian fibroblasts. The reason why hydrodynamic pressure increased transduction efficiency is still unclear, however we hypothesize that temporary stretching of the cell under hydrodynamic stress increases cell surface area and consequently provides an increased surface area for interaction between the cell membrane and viral particles.
The evidence presented in this study indicates that minor modifications such as altering the viral exposure conditions and an additional centrifugation step during exposure to the reprogramming factors can significantly improve the reprogramming efficiency, even in primary fibroblasts isolated from centenarian subjects which have low proliferative rate. Successful and relatively efficient reprogramming of centenarian somatic cells may allow the use of these cells in future regenerative medicine applications and tissue repair if the safety of the cell and tissue products can be established. The use of cellular models with reprogrammed and unreprogrammed cells and cells and tissues generated from the differentiation of the reprogrammed centenarian somatic cells will provide invaluable tools for the study of aging and longevity.
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Edited by Engadin, 30 April 2019 - 04:26 PM.
