Skip to main content
Download PDF

Xeno-free generation of human induced pluripotent stem cells from donor-matched fibroblasts isolated from dermal and oral tissues

Stem Cell Research & Therapy volume 14, Article number: 199 (2023) Cite this article

Abstract

Background

Induced pluripotent stem cells (iPS) can be generated from various somatic cells and can subsequently be differentiated to multiple cell types of the body. This makes them highly promising for cellular therapy in regenerative medicine. However, to facilitate their clinical use and to ensure safety, iPS culturing protocols must be compliant with good manufacturing practice guidelines and devoid of xenogenic products. Therefore, we aimed to compare the efficiency of using humanized culture conditions, specifically human platelet lysate to fetal bovine serum, for iPS generation from different sources, and to evaluate their stemness.

Methods

iPS were generated via a platelet lysate or fetal bovine serum-based culturing protocol from matched dermal, buccal and gingival human fibroblasts, isolated from healthy donors (n = 2) after informed consent, via episomal plasmid transfection. Pluripotency, genotype and phenotype of iPS, generated by both protocols, were then assessed by various methods.

Results

More attempts were generally required to successfully reprogram xeno-free fibroblasts to iPS, as compared to xenogenic cultured fibroblasts. Furthermore, oral fibroblasts generally required more attempts for successful iPS generation as opposed to dermal fibroblasts. Morphologically, all iPS generated from fibroblasts formed tight colonies surrounded by a reflective “whitish” outer rim, typical for iPS. They also expressed pluripotency markers at both gene (SOX2, OCT4, NANOG) and protein level (SOX2, OCT4). Upon stimulation, all iPS showed ability to differentiate into the three primary germ layers via expression of lineage-specific markers for mesoderm (MESP1, OSR1, HOPX), endoderm (GATA4) and ectoderm (PAX6, RAX). Genome analysis revealed several amplifications and deletions within the chromosomes of each iPS type.

Conclusions

The xeno-free protocol had a lower reprogramming efficiency compared to the standard xenogenic protocol. The oral fibroblasts generally proved to be more difficult to reprogram than dermal fibroblasts. Xeno-free dermal, buccal and gingival fibroblasts can successfully generate iPS with a comparable genotype/phenotype to their xenogenic counterparts.

Introduction

Mesenchymal stem cells (MSC) have long been instrumental in regenerative medicine due to their multipotency, ability to self-renew and high proliferative capacity [1,2,3]. Promising results utilizing MSC for bone regeneration in both preclinical and clinical settings have been reported recently [4,5,6,7,8,9]. However, due to a number of challenges surrounding MSC, the quest to exploring alternative sources, such as pluripotent cells, is crucial [10,11,12]. Embryonic stem cells (ESC) can be expanded indefinitely without undergoing replicative senescence or aging due to their high telomerase expression. The main property that sets ESC apart from other cells is their pluripotent nature, meaning they can give rise/differentiate to cells of the three primary germ layers (mesoderm, endoderm and ectoderm) [13]. With time, however, concerns were raised surrounding the use of ESC due to drawbacks associated with the isolation process, immunogenicity and risk of teratoma formation [13,14,15]. The discovery of induced pluripotent stem cells (iPS), artificially generated via genetic alteration/reprogramming of mature somatic cells, offered some advantages relative to ESC [16,17,18]. Similar to ESC, iPS express pluripotent markers, have unlimited proliferation potential and possess the ability to differentiate into the three primary germ layers [19, 20]. Furthermore, iPS can be generated autologously and/or from a selected genetic background [15, 19]. To gain pluripotency, the reprogramming process typically involves transfecting adult somatic cells with certain pluripotency markers. Yamanaka initially reprogrammed fibroblasts by using four transcription factors, OCT4, SOX2, KLF4 and c-MYC, also known as the OSKM factors, or the Yamanaka factors [16, 17]. Since then, other groups have successfully reprogrammed adult cells to iPS via the use of various different cocktails of transcription factors [18, 21]. A multitude of methods for delivering these factors have been developed, which include both integrating and non-integrating methods. Integrating methods, such as retro and lentiviral delivery methods, leave behind an undesirable footprint via integration of exogenous genetic material into the cell genome. Therefore, in order to make iPS more clinically applicable, non-integrating reprogramming methods have been developed with zero footprint [21, 22]. These non-integrating methods include, among others, the use of episomal plasmids for delivery. Studies have shown that the episomal vectors do not persist and are spontaneously lost after a period of time [21, 23]. Nevertheless, despite the absence of any signs of the original plasmids, other footprints are possible, including artificially introduced genetic alterations. Copy number variations (CNV), such as deletions, insertions and duplications, exist naturally as structural variants in the human genome [24]. Such sub-chromosomal aberrations, along with whole-chromosome aneuploidies, have been regularly reported in human pluripotent cells (both ESC and iPS), with iPS likely having a higher number of CNVs than ESC [25,26,27]. This could be due to the reprogramming process itself being associated with increased CNV levels in the early stages of the resultant iPS. The number and total size of these CNVs, however, decrease dramatically with continued propagation/passaging of iPS [28]. Fibroblasts, specifically dermal fibroblasts, were the first cell type to be utilized for iPS generation [17]. Since then, different somatic cells have been used to generate iPS including keratinocytes, blood cells, dental pulp stem cells and mesenchymal stem cells [29,30,31,32,33]. Fibroblasts remain the most commonly used cell type for iPS generation, as they are generally easy to obtain and handle, and are commercially available for research purposes. Various cell types from the oral cavity have been used for the generation of iPS, as cells can be easily collected during dental procedures, without the need for extra invasiveness. In addition, wounds in the oral cavity heal rapidly without scar formation and with minimal patient discomfort [34, 35]. Hence, transitioning to oral sources for iPS generation has the potential to be a valuable approach.

To comply with good manufacturing practice (GMP) guidelines, it is vital to create cell culture protocols that are safe and standardized. Currently, media supplemented with fetal bovine serum (FBS) is the most widely used method of cell expansion [36]. Despite its large scale and frequent use, FBS is associated with a multitude of ethical, scientific and safety issues. For instance, variations in serum composition result in batch-to-batch heterogeneity, causing morphological and phenotypical differences, ultimately affecting reproducibility of cell expansion protocols [37,38,39]. As a result, FBS production has come under great scrutiny, and there is an increasing demand and need for animal-free culture techniques, which would allow for a safer and more ethical practice. With that being said, xeno-free alternatives have been developed, and platelet lysate (PL) has emerged as a promising “GMP compliant” candidate to replace FBS. PL is typically prepared from platelet derivatives which contain and release high concentrations of growth factors. These growth factors aid in the expansion of cells in culture. Moreover, PL is generally pooled from multiple donors, reducing donor-based variations [40, 41]. These attributes make PL an attractive alternative to FBS as a supplement to expansion media.

A few studies have claimed to generate iPS from xeno-free conditions, yet their protocol either includes FBS media for the culture of the somatic cells prior to iPS generation, or they fail to mention the somatic cell culture method altogether [42,43,44]. As far as we know, only a handful of studies have previously generated iPS using an entirely xeno-free protocol [45,46,47,48]. However, no effort was made to compare iPS generated from xeno-free protocols to those generated from xenogenic ones. Due to the aforementioned challenges, it is important to implement protocols that comply with GMP guidelines, i.e., xeno-free culturing protocols, while also utilizing easily accessible sources for iPS generation. Therefore, in this study we aimed to analyze and compare iPS generated from donor-matched fibroblasts from different sources and evaluate their stemness. Furthermore, the study was aimed at evaluating and comparing the efficiency of using humanized culture conditions, specifically PL to FBS, on the generation of potent iPS.

Materials and methods

Fibroblast source and culture

Dermal, buccal and gingival samples were acquired from two healthy voluntary donors (Donor 1 (D1); female aged 40–50, and donor 2 (D2); female aged 50–60), following informed consent. All three samples were collected from each donor; dermal samples were obtained from the anterior forearm, buccal samples from the inside of the cheek and gingival samples from the gingiva above the upper first molar. The specimens were transported in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, ThermoFisher Scientific, Massachusetts, USA) supplemented with 3% penicillin/streptomycin (GE, Healthcare) and immediately processed for fibroblast isolation. Briefly, fibroblasts were isolated via the enzymatic digestion protocol as previously described [49]. Following isolation, fibroblasts were cultured and expanded in DMEM supplemented with 5% PL (Blood Bank, Haukeland University Hospital, Bergen, Norway) or 10% FBS (Gibco, ThermoFisher Scientific, Massachusetts, USA, catalog number: 10270106), creating two different culture conditions for cell expansion. The cells cultured in FBS were supplemented with 1% penicillin/streptomycin (GE, Healthcare), while the cells in PL were supplemented with 1% penicillin/streptomycin and heparin at a concentration of 2 IU/ ml (LEO Pharma). The morphology of the cells was observed using Nikon’s Inverted Light Microscope ECLiPSE Ts2R-FL (NIKON INSTRUMENTS EUROPE B.V., Amsterdam, the Netherlands). The fibroblast cell lines were regularly checked for mycoplasma contamination, and all lines were free of contamination prior to transfection.

Fibroblast reprogramming and iPS culture

Approximately 5—6 × 105 cells (passage 7–10) were transfected (Nucleofector 2b Device, Lonza, Switzerland) with 1 µg of each of the three episomal reprogramming plasmids (pCXLE-hOCT3/4-shp53, OCT4 & shRNA p53; pCXLE-kSK, SOX2 & KLF 4; pCXLE hUL, L-MYC & LIN28) and plated onto a six-well plate containing either FBS or PL supplemented DMEM (Gibco, ThermoFisher Scientific). Upon reaching confluency, the cells were passaged onto a 10 cm dish precoated with Geltrex (Gibco, ThermoFisher Scientific). The following day, culture media was changed to StemFlex media (StemFlex Medium, Gibco, ThermoFisher Scientific). Media was then changed every 1–2 days until stable colonies began to appear. Colonies were deemed stable upon formation of compact, round colonies with distinct borders [50]. Three colonies were then transferred to a Geltrex (Gibco, ThermoFisher Scientific) coated well in a 24-well plate, one colony per well. Each iPS colony was cultured individually in StemFlex media (StemFlex Medium, Gibco) and considered to be a biological replica. Gentle Cell Dissociation Reagent (Stem Cell Technologies, Vancouver, Canada) was used for cell detachment for passaging. Characterization and analyses of iPS were performed after passage 15. Cell morphology was observed using Nikon’s Inverted Light Microscope ECLiPSE Ts2R-FL (NIKON INSTRUMENTS EUROPE B.V., Amsterdam, the Netherlands). The iPS were regularly checked for mycoplasma contamination, and all lines were free of contamination prior to analysis.

iPS characterization

Trilineage differentiation

The iPS were subjected to directed differentiation using STEMdiff Trilineage Differentiation Kit (Stem Cell Technologies, Vancouver, Canada). Briefly, iPS were detached from well plates using Gentle Cell Dissociation Reagent (Stem Cell Technologies) and centrifuged at 300 g for 5 min. The pellet was then resuspended in 1 ml of Single Cell Plating Medium according to the manufacturer’s instructions. The cells (8 × 105 cells/well for endoderm and ectoderm differentiation and 2 × 105/well for mesoderm differentiation) were transferred to a Geltrex coated 12-well plate. The media was then changed daily for 5 days (mesoderm and endoderm) and 7 days (ectoderm).

iPS Gene expression analysis (RT-PCR)

The pluripotency of the iPS along with their ability to differentiate into the three primary germ layers was assessed via quantitative real-time PCR (qPCR). Total RNA was isolated using a tissue RNA isolation kit (Maxwell, Promega, WI, USA), and a NanoDrop spectrophotometer (ThermoScientific, Delaware, USA) was used to check the quantity and purity of the isolated RNA. Total RNA (300 ng) was reverse transcribed, according to the manufacturer’s instructions, using a high-capacity complementary DNA reverse transcription kit (Applied Biosystems, CA, USA). qPCR was performed on a StepOne Plus system, using TaqMan gene expression assays (Applied Biosystems), to quantify the gene expression of pluripotency markers (SOX2, OCT4, NANOG) and trilineage markers for mesoderm (MESP1, OSR1, HOPX), endoderm (GATA4) and ectoderm (PAX6, RAX) lineages. Data were analyzed using the ∆∆Ct method. Gene expression was normalized to that of the housekeeping gene, GAPDH. Expression of pluripotency markers is presented as fold changes relative to the control, dermal fibroblasts (DF) in FBS. Expression of trilineage markers is presented as fold changes relative to uninduced DF-iPS in FBS. An overview of the primers used for the gene expression analysis is presented in Additional file 1.

Flow cytometry

The iPS phenotype was analyzed via flow cytometry for specific markers, namely SOX2 and OCT4 (BD Biosciences, San Jose, CA, USA), according to the manufacturer’s instructions. The cells (~ 5 × 105) were fixed in 10% buffered formalin, for 10 min, and permeabilized via 0.1% Triton X, 15 min in the dark. The pellet was then blocked in 0.5% bovine serum albumin, BSA (Sigma-Aldrich, St. Louis, MO, USA), in phosphate-buffered saline (PBS), for 10 min at room temperature. Conjugated monoclonal antibodies were then added to the pellet, and the cells were incubated in the dark for 30 min at 4 °C. The cells were then washed with PBS before being resuspended in PBS. Stained samples were analyzed and compared to the corresponding unstained samples (negative control). The final quantification was performed with a BD Accuri flow cytometer (BD Biosciences), and the data were analyzed using FlowJo (FlowJo, LLC, Ashland, OR, USA).

Chromosome microarray analysis

Whole-genome high-resolution chromosome microarray analysis was performed using the Applied Biosystems CytoScan HD Array Kit and Reagent Kit Bundle (Applied Biosystems Catalog number: 901835) according to the manufacturer's protocol. Briefly, 250 ng of genomic DNA was digested with the restriction enzyme NspI and then ligated to an adapter, followed by PCR amplification using a single pair of adapter primers. The PCR products were purified using magnetic beads (Agencourt AMPure, Beckman Coulter, Beverly, MA). Purified PCR products were then fragmented using DNase I, and the fragmented PCR products end-labeled with biotin and then hybridized to the array using the Affymetrix GeneChip Hybridization Oven 645 (Affymetrix Inc., USA). Arrays were washed and stained using a GeneChip Fluidics Station 250 and scanned using an Affymetrix GeneChip Scanner 3000 7G (Affymetrix Inc.). Scanned data files were generated using Affymetrix GeneChip Command Console Software, version 4.1, and analyzed with Affymetrix Chromosome Analysis Suite version 4.2.1 (ChAS) (Affymetrix Inc.) and BENCH Lab CNV—version 5.1.12 (Agilent Technologies, USA). Filtration was performed against a list of common abbreviations acquired from Affymetrix. Duplications were filtered if at least 90% overlap, containing at least 80 markers and listed at least 25 times in the list of common abbreviations. Deletions were filtered if 90% overlap, containing at least 30 markers and listed at least 25 times in the list of common abbreviations. LSCH regions less than 5 Mbp or supported by less than 500 markers were filtered.

Ethical approval

Approval was granted by the Ethical Committee for Medical and Health Related Research in West Norway (REK 80005). Tissue samples were collected from two healthy voluntary donors after obtaining informed consent.

Data presentation and statistical analysis

Statistical analysis was performed via IBM SPSS Statistics (SPSS Inc.). Data are presented as mean values (± standard deviation). Statistical significance was determined via an independent samples T-test when comparing two groups and one-way analysis of variance (ANOVA) when comparing more than two groups. A p-value of < 0.05 was considered statistically significant.

Results

Isolation of fibroblasts in xeno-free conditions

The isolated matched dermal, buccal (BF) and gingival (GF) fibroblasts displayed a spindle-shaped morphology (Fig. 1).

Fig. 1
figure 1

Representative light microscopy images demonstrating the morphology of the fibroblasts from different sources expanded in FBS (A–C) and PL (D–F). Scale bar: 100 µm (DF: dermal fibroblasts, BF: buccal fibroblasts, GF: gingival fibroblasts, FBS: fetal bovine serum, PL: platelet lysate)

Full size image

Generation of xeno-free iPS

Matched DF, BF and GF from 2 donors were reprogrammed into iPS. The protocols for generating iPS from the different fibroblasts are illustrated in Fig. 2. The maximum number of reprogramming attempts was set at nine, after which the success rate (based on the number of attempts required to successfully develop iPS colonies) was determined. All the iPS reprogrammed successfully with the exception of one sample of gingival fibroblasts isolated in PL (GF-PL, D2). Fibroblasts isolated and grown in FBS generally showed a higher reprogramming success rate than those in PL. In most cases, DF showed the highest reprogramming success rate, while BF and GF showed varying success rates with no clear pattern (Table 1). Due to the BF-PL from D2 not yielding any iPS, the characterization of the iPS in this study was limited to the those obtained from D1.

Fig. 2
figure 2

A Diagram illustrating the xeno/xeno-free generation of iPS, via episomal plasmid transfection, from different sources of matched fibroblasts. B Detailed illustration of the transfection/reprogramming procedure. This figure was created using Procreate 5.2 on iOS software (iPS: induced pluripotent stem cells, FBS: fetal bovine serum, PL: platelet lysate)

Full size image
Table 1 Data on the reprogramming of fibroblasts expanded in FBS/PL supplemented media
Full size table

Stemness of the generated iPS

All the reprogrammed fibroblasts developed a stable colony morphology resembling ESC [50]. Morphologically, the cells grew in colonies, surrounded by a reflective “whitish” border, that increased in size as the cells proliferated, ultimately fusing with other colonies in the same well (Fig. 3).

Fig. 3
figure 3

Representative light microscopy images demonstrating the morphology of the iPS from different sources expanded in FBS (A–C) and PL (D–F). Scale bar: 200 µm (DF: dermal fibroblasts, BF: buccal fibroblasts, GF: gingival fibroblasts, iPS: induced pluripotent stem cells, FBS: fetal bovine serum, PL: platelet lysate)

Full size image

Generated iPS express pluripotent genes

Gene expression analysis showed that the iPS expressed significantly higher levels (p < 0.001) of the pluripotency markers, SOX2, OCT4 and NANOG, than their respective controls (undifferentiated fibroblasts) which showed little to no expression of these markers (Fig. 4). The iPS-FBS generally displayed higher levels of these pluripotency markers than their matched iPS-PL (Fig. 4).

Fig. 4
figure 4

Relative gene expression of A SOX2 B OCT4 and C) NANOG by iPS in FBS/PL. Expression is presented relative to the DF-iPS-FBS group ± SD. D Heatmap analysis of the gene expression of SOX2, OCT4 and NANOG by the fibroblasts in FBS/PL and their resultant iPS. Expression presented relative to the DF-FBS group. Independent samples t-test and one-way ANOVA were used to determine statistical significance (p < 0.05). (*) represents significance between iPS, from the same source, grown in FBS to those grown in PL. (+) represents significance between the iPS-FBS. (#) represents significance between the iPS-PL (DF: dermal fibroblasts, BF: buccal fibroblasts, GF: gingival fibroblasts, iPS: induced pluripotent stem cells, FBS: fetal bovine serum, PL: platelet lysate)

Full size image

Within the iPS-FBS, the GF-iPS revealed higher expression of SOX2 and NANOG than both BF-iPS (significantly higher) and DF-iPS (significantly higher in the case of NANOG). Expression of OCT4 by the iPS-FBS was comparable. The DF-iPS in turn revealed higher expression of SOX2 (significantly higher) and NANOG than the BF-iPS, and slightly higher expression of OCT4 than both the oral iPS. Within the iPS-PL, the GF-iPS showed a significantly higher expression of OCT4 and NANOG than both the DF-iPS and BF-iPS, which in turn showed comparable expression. OCT4 expression was also significantly higher in the BF-iPS when compared to the DF-iPS. SOX2 expression was significantly higher in the DF-iPS than both the BF-iPS and GF-iPS (comparable).

Proteomics of pluripotency markers

Flow cytometric analysis generally revealed SOX2 and OCT4 positive iPS-FBS and iPS-PL. Analysis of the iPS-FBS revealed > 97% of cells positive for SOX2 and > 91% positive for OCT4. Analysis of the iPS-PL revealed > 97% of cells positive for SOX2 and > 94% positive for OCT4 (Fig. 5).

Fig. 5
figure 5

Flow cytometric analysis of A iPS-FBS and B iPS-PL showing detection of intracellular pluripotent markers SOX2 and OCT4 (percentage averages ± standard deviation) (iPS: induced pluripotent stem cells, FBS: fetal bovine serum, PL: platelet lysate)

Full size image

Trilineage differentiation ability of iPS

Upon stimulation, all iPS showed ability to differentiate into the three germ layers, via expression of lineage-specific markers for mesoderm (MESP1, OSR1, HOPX) (Fig. 6A–C), endoderm (GATA4) (Fig. 6D) and ectoderm (PAX6, RAX) markers (Fig. 6E, F). Induced iPS showed significantly (p < 0.05) higher expression of the trilineage markers than their respective controls (undifferentiated iPS).

Fig. 6
figure 6

Relative gene expression of the A–C mesoderm, D endoderm and E, F ectoderm markers by the iPS following directed differentiation toward the three lineages. G Heatmap analysis of the gene expression of trilineage markers by the iPS before and after directed (induced) differentiation. Expression is presented relative to the uninduced DF-iPS-FBS group ± SD (uninduced iPS not shown on graphs). Independent samples t-test and one-way ANOVA were used to determine statistical significance (p < 0.05). (*) represents significance between the induced iPS, from the same source, grown in FBS to those grown in PL. ( +) represents significance between the induced iPS-FBS. (#) represents significance between the induced iPS-PL (DF: dermal fibroblasts, BF: buccal fibroblasts, GF: gingival fibroblasts, iPS: induced pluripotent stem cells, FBS: fetal bovine serum, PL: platelet lysate, ENDO: endoderm, MESO: mesoderm, ECTO: ectoderm)

Full size image

Mesoderm

Induced iPS-PL expressed MESP1 at a significantly higher rate than their matched FBS, except in the case of DF-iPS where the expression was comparable. Expression of OSR1 was comparable between the induced iPS-FBS and iPS-PL. Induced iPS-FBS revealed significantly higher expression of HOPX than their respective matched iPS-PL, except in the case of the BF-iPS where the iPS-PL displayed significantly higher expression than the iPS-FBS.

Within the induced iPS-FBS, expression of mesoderm markers was generally comparable throughout. Within the induced iPS-PL, MESP1 expression was higher in the oral iPS than the DF-iPS, while HOPX expression by the DF-iPS and BF-iPS was significantly higher than the GF-iPS. The expression of OSR1 was comparable in the iPS-PL.

Endoderm

Induced iPS-FBS expressed significantly higher levels of GATA4 than their matched iPS-PL, except in the case of the DF-iPS where expression was slightly higher in the iPS-PL. Within the induced iPS-FBS, the BF-iPS expressed higher level of GATA4 than both the DF-iPS and the GF-iPS (significantly higher), with the DF-iPS expressing higher levels than the GF-iPS. In the PL group, the DF-iPS showed higher GATA4 expression than both the BF-iPS and the GF-iPS (significantly higher), with the BF-iPS expressing higher levels than the GF-iPS.

Ectoderm

Induced iPS-FBS expressed higher levels of PAX6 than their matched iPS-PL (significantly higher in the case of the GF-iPS), except in the case of BF-iPS where expression was comparable. RAX expression was higher in the iPS-FBS than their matched iPS-PL (significantly higher in the case of GF-iPS), except in the case of the DF-iPS where the iPS-PL displayed significantly higher expression levels.

Within the iPS-FBS, PAX6 expression was comparable. The DF-iPS and GF-iPS showed comparable expression levels and significantly higher levels than the BF-iPS. Within the induced iPS-PL, PAX6 expression by the GF-iPS was downregulated compared to the DF-iPS and BF-iPS, which showed comparable expression. RAX expression was highest in the DF-iPS, followed by the BF-iPS.

Genetic stability of iPS

Chromosomal analysis revealed multiple amplifications and deletions within the genome of the iPS (Fig. 7). The iPS-FBS showed amplifications in chromosome 1, 5, 13 and X, and deletions in chromosomes 4, 11 and 16. The iPS-PL showed amplifications in chromosomes 1, 5, 6, 8, 13 and 17, and deletions in chromosomes 7, 11 and 16. A detailed genomic analysis of the iPS, including size and locations of the CNVs, can be found in Additional file 2.

Fig. 7
figure 7

Representative* figure displaying the chromosomal CNVs of the different iPS-FBS and iPS-PL. The arrows/bars represent the gain (blue) or loss (red) of a chromosomal region. *Intended only as a representative figure and not for displaying exact locations of each CNV (DF: dermal fibroblasts, BF: buccal fibroblasts, GF: gingival fibroblasts, iPS: induced pluripotent stem cells, FBS: fetal bovine serum, PL: platelet lysate)

Full size image

Discussion

To comply with GMP guidelines, cells must be cultured in xeno-free conditions prior to their use for iPS generation. To our knowledge, only a handful of studies have generated iPS from entirely xeno-free conditions [45,46,47,48]. Additionally, in most cases, no effort was made to compare iPS generated via xeno-free protocols to those generated in xenogenic ones. This paper is strengthened by the standardized conditions that the cells were subjected to throughout the entirety of the project. Donor-matched fibroblasts were cultured in xeno-free PL supplemented media and separately in xenogenic FBS supplemented media, simultaneously, from the time of isolation up until 1 week post-transfection. In addition, the fibroblasts were all transfected with the same cocktail of transcription factors, via the same method of delivery and in the same laboratory. These standardized conditions allow for efficient comparisons between xenogenic and xeno-free fibroblasts in the generation of iPS.

In this study, the reprogramming of donor-matched DF, BF and GF from two donors was attempted. The attempts, however, were not always successful, as presented in Table 1. Chow et al. also reported similar difficulties in obtaining iPS colonies from adult canine DF [51]. Such difficulties generally represent one of the major drawbacks associated with iPS production, as the efficiency of reprogramming somatic cells is deemed to be quite low, with efficiency levels as low as 0.0006% [22]. Several theories have been postulated in attempts to explain why only a small portion of transduced cells gain pluripotency. The general consensus is that cell reprogramming comprises two main phases: a primary stochastic phase and a secondary more deterministic phase [20]. Completion of both phases appears to be a rare event for most cells, hence the low reprogramming efficiency levels. With that being said, it is reasonable to expect that in some cases the reprogramming cycle would fail altogether. From the data presented here, it seems that a transition to a PL culturing protocol was less supportive of fibroblast reprogramming to iPS. A previous report from Sung et al. corroborates our results, where in their study, PL was also found to be less efficient at inducing cell reprogramming of human amniotic fluid stem cells. Reprogramming efficiency was also found to be significantly higher in the cells cultured in FBS supplemented media [46]. The literature has shown that PL culturing protocols do indeed affect the behavior of other cell types as well. For instance, PL has been reported to increase fibroblast proliferation rates compared to FBS [52]. In MSC, both disruption and maintenance of the undifferentiated cell state have been reported to be induced by PL [7, 53,54,55]. These conflicting reports could be attributed to the heterogeneity that exists between PL batches, due to the pooling of the blood derivatives from multiple donors [56]. Such PL-associated changes may likely have an effect on the behavior of fibroblasts and in turn affect their ability to differentiate to iPS.

When comparing cells from multiple individuals, despite the cell type being the same, donor variability must be accounted for. Cells obtained from different individuals tend to behave differently, both morphologically and functionally [57]. Similarly, inter-donor disparities, among other factors, may lead to variations among iPS [50, 58, 59]. For instance, cells obtained from the elderly are associated with an increased risk of iPS abnormalities and a decrease in reprogramming efficiency [60,61,62]. The results from this study clearly show different cellular responses, between the two donors, to the transfection procedure. For example, the BF from D1 (FBS/PL—100% success rate) were highly susceptible to reprogramming compared to the BF from D2 (FBS—33% success rate, PL—0% success rate). Besides donor variation, such differences may also be attributed to CNVs that might be acquired during the fibroblast reprogramming process [63]. These findings advocate for further investigations on the effect of donor variability on cellular reprogramming/iPS generation.

Certain elements must be considered when attempting to select the optimal cell source for reprogramming purposes, including invasiveness of the surgical procedure, ease of isolation and maintenance, and susceptibility to the reprogramming process [64]. Different somatic cell types have displayed varying results in terms of reprogramming susceptibility. Studies have revealed, for example, that keratinocytes are more easily reprogrammed than fibroblasts and that dental pulp stem cells yield more iPS colonies than bone marrow MSC [30, 33]. This disparity in susceptibility makes selection of the ideal cell source for reprogramming quite difficult. In this study, we found that DF are generally easier to reprogram than BF and GF. Yan et al. also reported similar difficulties when attempting to reprogram GF, with various transfection protocols yielding no iPS colonies [33]. It is not clear in their study, however, how many attempts were made to generate iPS from the GF. As it is with the GF in this study, it might be that continued attempts would have eventually led to the development of pluripotent colonies. The exact reasons as to why oral fibroblasts reprogram less efficiently than DF are unclear. However, inherent phenotypical differences between the fibroblasts are likely to play a role in reprogramming efficiency [65, 66]. Further investigation is required to determine the correlation and effect such innate characteristics have on the reprogramming process.

Due the BF-PL from D2 not yielding any iPS colonies, the focus was shifted to the results obtained from the analysis of D1. This allows for a more efficient comparison of the effect that different sources and culture conditions have on iPS generation. According to the literature, there are different levels of pluripotency, and cells should fulfill certain criteria at each level before being deemed as pluripotent [67, 68]. These criteria include cell/colony morphology, expression of pluripotency markers and ability to differentiate into the three primary germ layers. Once the iPS in this study were established, we analyzed their pluripotency at a cellular, molecular and functional level. At a cellular level, all the cells developed a similar colony morphology to ESC, and although not identical, they fall in the category of stable iPS colony morphology [50, 69]. At a molecular level, they expressed genes (SOX2, OCT4, NANOG) and proteins (OCT4, SOX2) which play a major role in inducing and maintaining the pluripotency of ESC and iPS [68]. They also displayed functional pluripotency and expressed markers associated with mesoderm (MESP1, OSR1, HOPX), endoderm (GATA4) and ectoderm (PAX6, RAX) lineages following directed differentiation. These findings demonstrate that these iPS are pluripotent and possess ESC-like characteristics. Despite the decrease in reprogramming efficiency, moving to a xeno-free protocol does not seem to have any detrimental effect on the cells after successful induction of pluripotency, as no major differences were seen between the genotype/phenotype of the iPS-PL and iPS-FBS [46]. At gene level, iPS-PL generally expressed slightly lower levels of the pluripotent markers than iPS-FBS; however, differences were insignificant. Furthermore, these differences did not seem to translate at protein level, with flow cytometry analysis revealing comparable detection of pluripotent proteins by both sets of iPS. The ability of iPS to differentiate to the three primary germ layers does not appear to be negatively affected by the use of PL, with the iPS-PL and iPS-FBS expressing comparable levels of the trilineage markers upon directed differentiation. The differentiated GF-iPS-PL expressed the markers GATA4 and RAX significantly higher than the non-differentiated iPS. However, this expression was much less than the rest of the differentiated iPS, including its xenogenic counterpart. The reason for this relatively low expression is unclear. Perhaps the xeno-free protocol caused these particular cells to differentiate much slower toward endoderm and ectoderm lineages, and an increase in the duration of differentiation might result in similar expression levels to the other iPS.

With the introduction of new supplements for cell culturing protocols, it is important to ensure that no major alterations occur within the cell genome as a result of supplementation. When assessing the genetic state of human pluripotent cells, the literature shows that chromosomes 1, 12, 17, 20 and X are generally the most affected [26, 27]. Interestingly, a different set of chromosomes were more commonly affected within both iPS groups, specifically chromosomes 1, 5, 11 and 13. CNVs affecting these specific chromosomes were seen in all the iPS apart from BF-iPS-PL. Overall, both sets of iPS revealed a relatively low amount of CNVs, with the exception of the DF-iPS in both conditions, which show duplications of several segments in chromosome 1. Somatic mosaicism in the culture of fibroblasts has been shown to cause most of the genetic variation in their resultant iPS [26]. Similarly, Abyzov et al. [70] revealed in a study involving dermal fibroblasts/iPS, that 50% of the CNVs found in the iPS were present in their parental fibroblasts. This, however, is not the case with the DF-iPS in this study, as the genetic analysis revealed no CNVs in chromosome 1 of their parent fibroblasts. Hence, this particular genetic change is likely a result of the reprogramming process, or cell culture and propagation [71]. Despite their being links between aberrations in chromosome 1 to tumor formation [72], the majority of the evidence points to the harmlessness of chromosomal abnormalities in iPS. Ultimately, these genetic alterations are a common occurrence, as human pluripotent cells are often genetically unstable. Furthermore, both genomically normal and abnormal iPS can lead to teratoma formation, and there is little evidence linking the genomic abnormalities with tumorigenesis [26, 27, 63].

Analyzing the effect of cell source on iPS genotype/phenotype revealed only minor differences within each group. No particular trend was observed, however, and thus, none of the variations could be directly attributed to differences in cell source. Ultimately, these results confirm the ability to generate safe iPS from oral and dermal fibroblasts in xeno-free conditions, with quality comparable to those generated in FBS. This should allow for a smooth transition to utilizing xeno-free oral iPS for research at both the preclinical and clinical stage.

Conclusion

For the purposes of future stem cell research and clinical translation, generating iPS in xeno-free conditions serves as a favorable strategy. When compared to FBS, the use of PL in culture media appears to lower reprogramming efficiency. Nevertheless, xeno-free dermal, buccal and gingival fibroblasts can successfully generate iPS similar to their xenogenic counterparts. The nature of fibroblast source and expansion conditions appear to have little effect on iPS genotype/phenotype. Despite having the advantage of rapid healing with minimal scar formation, oral fibroblasts proved to be more difficult to reprogram than dermal fibroblasts. Transitioning to xeno-free oral fibroblasts for generating iPS looks to be a promising approach; however, the issue of low reprogramming efficiency must be addressed in order to boost cost-effectiveness for future research and clinical use.

Availability of data and materials

Additional data can be made available by the authors upon request. Chromosome microarray analysis data archived and shared, according to SN data policy, to NCBI’s dbVar public repository (accession: nstd231) at https://www.ncbi.nlm.nih.gov/dbvar/studies/nstd231/.

Abbreviations

BF:

Buccal fibroblasts

CNV:

Copy number variation

D:

Donor

DF:

Dermal fibroblasts

DMEM:

Dulbecco’s modified Eagle’s medium

ESC:

Embryonic stem cells

FBS:

Fetal bovine serum

GF:

Gingival fibroblasts

GMP:

Good manufacturing practice

iPS:

Induced pluripotent stem cells

MSC:

Mesenchymal stem cells

PL:

Platelet lysate

References

  1. Langer R, Vacanti JP. Tissue engineering. Science. 1993;260(5110):920–6.

    Article  CAS  PubMed  Google Scholar 

  2. Gimble Jeffrey M, Katz Adam J, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res. 2007;100(9):1249–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284(5411):143–7.

    Article  CAS  PubMed  Google Scholar 

  4. Gjerde C, Mustafa K, Hellem S, Rojewski M, Gjengedal H, Yassin MA, et al. Cell therapy induced regeneration of severely atrophied mandibular bone in a clinical trial. Stem Cell Res Ther. 2018;9(1):213.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Mohamed-Ahmed S, Fristad I, Lie SA, Suliman S, Mustafa K, Vindenes H, et al. Adipose-derived and bone marrow mesenchymal stem cells: a donor-matched comparison. Stem Cell Res Ther. 2018;9(1):1–15.

    Article  Google Scholar 

  6. Shanbhag S, Mohamed-Ahmed S, Lunde THF, Suliman S, Bolstad AI, Hervig T, et al. Influence of platelet storage time on human platelet lysates and platelet lysate-expanded mesenchymal stromal cells for bone tissue engineering. Stem Cell Res Ther. 2020;11(1):351.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Suliman S, Ali HR, Karlsen TA, Amiaud J, Mohamed-Ahmed S, Layrolle P, et al. Impact of humanised isolation and culture conditions on stemness and osteogenic potential of bone marrow derived mesenchymal stromal cells. Sci Rep. 2019;9(1):1–18.

    Article  CAS  Google Scholar 

  8. Gómez-Barrena E, Rosset P, Gebhard F, Hernigou P, Baldini N, Rouard H, et al. Feasibility and safety of treating non-unions in tibia, femur and humerus with autologous, expanded, bone marrow-derived mesenchymal stromal cells associated with biphasic calcium phosphate biomaterials in a multicentric, non-comparative trial. Biomaterials. 2019;196:100–8.

    Article  PubMed  Google Scholar 

  9. Gómez-Barrena E, Padilla-Eguiluz N, Rosset P, Gebhard F, Hernigou P, Baldini N, et al. Early efficacy evaluation of mesenchymal stromal cells (MSC) combined to biomaterials to treat long bone non-unions. Injury. 2020;51:S63–73.

    Article  PubMed  Google Scholar 

  10. Zhang J, Huang X, Wang H, Liu X, Zhang T, Wang Y, et al. The challenges and promises of allogeneic mesenchymal stem cells for use as a cell-based therapy. Stem Cell Res Ther. 2015;6:234.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Ikebe C, Suzuki K. Mesenchymal stem cells for regenerative therapy: optimization of cell preparation protocols. Biomed Res Int. 2014;2014:11.

    Article  Google Scholar 

  12. Gao F, Chiu SM, Motan DAL, Zhang Z, Chen L, Ji HL, et al. Mesenchymal stem cells and immunomodulation: current status and future prospects. Cell Death Dis. 2016;7:e2062.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Mountford J. Human embryonic stem cells: origins, characteristics and potential for regenerative therapy. Transfus Med. 2008;18(1):1–12.

    Article  CAS  PubMed  Google Scholar 

  14. Luk F, Eggenhofer E, Dahlke M, Hoogduijn M. The use of stem cells for treatment of diseases. Front. Young Minds. 2017;5(9):1–6.

  15. Bhartiya D, Nagvenkar P, Sriraman K, Shaikh A. An overview of pluripotent stem cells. Pluripotent Stem Cells: IntechOpen; 2013.

  16. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126(4):663–76.

    Article  CAS  PubMed  Google Scholar 

  17. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131(5):861–72.

    Article  CAS  PubMed  Google Scholar 

  18. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007;318(5858):1917–20.

    Article  CAS  PubMed  Google Scholar 

  19. Shi Y, Inoue H, Wu JC, Yamanaka S. Induced pluripotent stem cell technology: a decade of progress. Nat Rev Drug Discov. 2017;16(2):115.

    Article  CAS  PubMed  Google Scholar 

  20. Omole AE, Fakoya AOJ. Ten years of progress and promise of induced pluripotent stem cells: historical origins, characteristics, mechanisms, limitations, and potential applications. PeerJ. 2018;6:e4370.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Okita K, Matsumura Y, Sato Y, Okada A, Morizane A, Okamoto S, et al. A more efficient method to generate integration-free human iPS cells. Nat Methods. 2011;8:409.

    Article  CAS  PubMed  Google Scholar 

  22. Rao MS, Malik N. Assessing iPSC reprogramming methods for their suitability in translational medicine. J Cell Biochem. 2012;113(10):3061–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Schlaeger TM, Daheron L, Brickler TR, Entwisle S, Chan K, Cianci A, et al. A comparison of non-integrating reprogramming methods. Nat Biotechnol. 2015;33(1):58.

    Article  CAS  PubMed  Google Scholar 

  24. Redon R, Ishikawa S, Fitch KR, Feuk L, Perry GH, Andrews TD, et al. Global variation in copy number in the human genome. Nature. 2006;444(7118):444–54.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Närvä E, Autio R, Rahkonen N, Kong L, Harrison N, Kitsberg D, et al. High-resolution DNA analysis of human embryonic stem cell lines reveals culture-induced copy number changes and loss of heterozygosity. Nat Biotechnol. 2010;28(4):371–7.

    Article  PubMed  Google Scholar 

  26. Peterson SE, Loring JF. Genomic instability in pluripotent stem cells: implications for clinical applications. J Biol Chem. 2014;289(8):4578–84.

    Article  CAS  PubMed  Google Scholar 

  27. Baker D, Hirst Adam J, Gokhale Paul J, Juarez Miguel A, Williams S, Wheeler M, et al. Detecting genetic mosaicism in cultures of human pluripotent stem cells. Stem Cell Rep. 2016;7(5):998–1012.

    Article  CAS  Google Scholar 

  28. Hussein SM, Batada NN, Vuoristo S, Ching RW, Autio R, Närvä E, et al. Copy number variation and selection during reprogramming to pluripotency. Nature. 2011;471(7336):58–62.

    Article  CAS  PubMed  Google Scholar 

  29. Niibe K, Kawamura Y, Araki D, Morikawa S, Miura K, Suzuki S, et al. Purified mesenchymal stem cells are an efficient source for iPS cell induction. PLoS ONE. 2011;6(3):e17610.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Raab S, Klingenstein M, Liebau S, Linta L. A comparative view on human somatic cell sources for iPSC generation. Stem Cells Int 2014;2014.

  31. Miyoshi K, Tsuji D, Kudoh K, Satomura K, Muto T, Itoh K, et al. Generation of human induced pluripotent stem cells from oral mucosa. Stem Cells Int. 2010;110(3):345–50.

    CAS  Google Scholar 

  32. Wada N, Wang B, Lin NH, Laslett AL, Gronthos S, Bartold PM. Induced pluripotent stem cell lines derived from human gingival fibroblasts and periodontal ligament fibroblasts. J Periodontal Res. 2011;46(4):438–47.

    Article  CAS  PubMed  Google Scholar 

  33. Yan X, Qin H, Qu C, Tuan RS, Shi S, Huang GT-J. iPS cells reprogrammed from human mesenchymal-like stem/progenitor cells of dental tissue origin. Stem Cells Dev. 2010;19(4):469–80.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Szpaderska AM, Zuckerman JD, DiPietro LA. Differential injury responses in oral mucosal and cutaneous wounds. J Dent Res. 2003;82(8):621–6.

    Article  CAS  PubMed  Google Scholar 

  35. Iglesias-Bartolome R, Uchiyama A, Molinolo AA, Abusleme L, Brooks SR, Callejas-Valera JL, et al. Transcriptional signature primes human oral mucosa for rapid wound healing. Sci Transl Med. 2018;10(451):eaap8798.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Gstraunthaler G. Alternatives to the use of fetal bovine serum: serum-free cell culture. Altex. 2003;20(4):275–81.

    PubMed  Google Scholar 

  37. Mannello F, Tonti GA. Concise review: no breakthroughs for human mesenchymal and embryonic stem cell culture: conditioned medium, feeder layer, or feeder‐free; medium with fetal calf serum, human serum, or enriched plasma; serum‐free, serum replacement nonconditioned medium, or ad hoc formula? All that glitters is not gold! Stem Cells. 2007;25(7):1603–9.

    Article  CAS  PubMed  Google Scholar 

  38. Jochems CE, Van Der Valk JB, Stafleu FR, Baumans V. The use of fetal bovine serum: ethical or scientific problem? Altern Lab Anim. 2002;30(2):219–28.

    Article  CAS  PubMed  Google Scholar 

  39. Karnieli O, Friedner OM, Allickson JG, Zhang N, Jung S, Fiorentini D, et al. A consensus introduction to serum replacements and serum-free media for cellular therapies. Cytotherapy. 2017;19(2):155–69.

    Article  CAS  PubMed  Google Scholar 

  40. Shanbhag S, Stavropoulos A, Suliman S, Hervig T, Mustafa K. Efficacy of humanized mesenchymal stem cell cultures for bone tissue engineering: a systematic review with a focus on platelet derivatives. Tissue Eng Part B Rev. 2017;23(6):552–69.

    Article  CAS  PubMed  Google Scholar 

  41. Astori G, Amati E, Bambi F, Bernardi M, Chieregato K, Schäfer R, et al. Platelet lysate as a substitute for animal serum for the ex-vivo expansion of mesenchymal stem/stromal cells: present and future. Stem Cell Res Ther. 2016;7(1):1–8.

    Article  Google Scholar 

  42. Totonchi M, Taei A, Seifinejad A, Tabebordbar M, Rassouli H, Farrokhi A, et al. Feeder-and serum-free establishment and expansion of human induced pluripotent stem cells. Int J Dev Biol. 2009;54(5):877–86.

    Article  Google Scholar 

  43. Rajala K, Lindroos B, Hussein SM, Lappalainen RS, Pekkanen-Mattila M, Inzunza J, et al. A defined and xeno-free culture method enabling the establishment of clinical-grade human embryonic, induced pluripotent and adipose stem cells. PLoS ONE. 2010;5(4):e10246.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Uhlin E, Rönnholm H, Day K, Kele M, Tammimies K, Bölte S, et al. Derivation of human iPS cell lines from monozygotic twins in defined and xeno free conditions. Stem Cell Res. 2017;18:22–5.

    Article  CAS  PubMed  Google Scholar 

  45. Rodríguez-Pizà I, Richaud-Patin Y, Vassena R, González F, Barrero MJ, Veiga A, et al. Reprogramming of human fibroblasts to induced pluripotent stem cells under xeno-free conditions. Stem Cells. 2010;28(1):36–44.

    Article  PubMed  Google Scholar 

  46. Sung T-C, Li H-F, Higuchi A, Kumar SS, Ling Q-D, Wu Y-W, et al. Effect of cell culture biomaterials for completely xeno-free generation of human induced pluripotent stem cells. Biomaterials. 2020;230:119638.

    Article  CAS  PubMed  Google Scholar 

  47. Lee K-I, Kim H-T, Hwang D-Y. Footprint- and xeno-free human iPSCs derived from urine cells using extracellular matrix-based culture conditions. Biomaterials. 2014;35(29):8330–8.

    Article  CAS  PubMed  Google Scholar 

  48. Wang J, Hao J, Bai D, Gu Q, Han W, Wang L, et al. Generation of clinical-grade human induced pluripotent stem cells in Xeno-free conditions. Stem Cell Res Ther. 2015;6(1):1–11.

    Article  Google Scholar 

  49. Costea DE, Kulasekara K, Neppelberg E, Johannessen AC, Vintermyr OK. Species-specific fibroblasts required for triggering invasiveness of partially transformed oral keratinocytes. Am J Pathol. 2006;168(6):1889–97.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Bjørlykke Y, Søviknes AM, Hoareau L, Vethe H, Mathisen AF, Chera S, et al. Reprogrammed cells display distinct proteomic signatures associated with colony morphology variability. Stem cells international. 2019;2019.

  51. Chow L, Johnson V, Regan D, Wheat W, Webb S, Koch P, et al. Safety and immune regulatory properties of canine induced pluripotent stem cell-derived mesenchymal stem cells. Stem Cell Res. 2017;25:221–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Fernández Muñoz B, Lopez-Navas L, Gonzalez Bermejo M, Lomas Romero IM, Montiel Aguilera Miguel Á, Campos Cuerva R, et al. A proprietary GMP human platelet lysate for the expansion of dermal fibroblasts for clinical applications. Platelets. 2021;1–12.

  53. Russell KA, Gibson TWG, Chong A, Co C, Koch TG. Canine platelet lysate is inferior to fetal bovine serum for the isolation and propagation of canine adipose tissue- and bone marrow-derived mesenchymal stromal cells. PLoS ONE. 2015;10(9):e0136621.

    Article  PubMed  PubMed Central  Google Scholar 

  54. Tsai C-C, Su P-F, Huang Y-F, Yew T-L, Hung S-C. Oct4 and nanog directly regulate Dnmt1 to maintain self-renewal and undifferentiated state in mesenchymal stem cells. Mol Cell. 2012;47(2):169–82.

    Article  CAS  PubMed  Google Scholar 

  55. Castiglia S, Mareschi K, Labanca L, Lucania G, Leone M, Sanavio F, et al. Inactivated human platelet lysate with psoralen: a new perspective for mesenchymal stromal cell production in good manufacturing practice conditions. Cytotherapy. 2014;16(6):750–63.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Burnouf T, Strunk D, Koh MBC, Schallmoser K. Human platelet lysate: Replacing fetal bovine serum as a gold standard for human cell propagation? Biomaterials. 2016;76:371–87.

    Article  CAS  PubMed  Google Scholar 

  57. Phinney DG, Kopen G, Righter W, Webster S, Tremain N, Prockop DJ. Donor variation in the growth properties and osteogenic potential of human marrow stromal cells. J Cell Biochem. 1999;75(3):424–36.

    Article  CAS  PubMed  Google Scholar 

  58. Vitale AM, Matigian NA, Ravishankar S, Bellette B, Wood SA, Wolvetang EJ, et al. Variability in the generation of induced pluripotent stem cells: importance for disease modeling. Stem Cells Transl Med. 2012;1(9):641–50.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Payne KA, Didiano DM, Chu CR. Donor sex and age influence the chondrogenic potential of human femoral bone marrow stem cells. Osteoarthr Cartil. 2010;18(5):705–13.

    Article  CAS  Google Scholar 

  60. Lo Sardo V, Ferguson W, Erikson GA, Topol EJ, Baldwin KK, Torkamani A. Influence of donor age on induced pluripotent stem cells. Nat Biotechnol. 2017;35(1):69–74.

    Article  CAS  PubMed  Google Scholar 

  61. Mahmoudi S, Brunet A. Aging and reprogramming: a two-way street. Curr Opin Cell Biol. 2012;24(6):744–56.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Trokovic R, Weltner J, Noisa P, Raivio T, Otonkoski T. Combined negative effect of donor age and time in culture on the reprogramming efficiency into induced pluripotent stem cells. Stem Cell Res. 2015;15(1):254–62.

    Article  PubMed  Google Scholar 

  63. Scholz H, Ræder H. Response to Comment on Carrasco et al. Spatial environment affects HNF4A mutation-specific proteome signatures and cellular morphology in hiPSC-derived β-like cells. Diabetes 2022;71:862–869. Diabetes. 2023;72(5):e3-e4.

  64. Maherali N, Hochedlinger K. Guidelines and techniques for the generation of induced pluripotent stem cells. Cell Stem Cell. 2008;3(6):595–605.

    Article  CAS  PubMed  Google Scholar 

  65. Lepekhin E, Grøn B, Berezin V, Bock E, Dabelsteen E. Differences in motility pattern between human buccal fibroblasts and periodontal and skin fibroblasts. Eur J Oral Sci. 2002;110(1):13–20.

    Article  PubMed  Google Scholar 

  66. Palaiologou AA, Yukna RA, Moses R, Lallier TE. Gingival, dermal, and periodontal ligament fibroblasts express different extracellular matrix receptors. J Periodontol. 2001;72(6):798–807.

    Article  CAS  PubMed  Google Scholar 

  67. Yilmaz A, Benvenisty N. Defining human pluripotency. Cell Stem Cell. 2019;25(1):9–22.

    Article  CAS  PubMed  Google Scholar 

  68. Smith KP, Luong MX, Stein GS. Pluripotency: toward a gold standard for human ES and iPS cells. J Cell Physiol. 2009;220(1):21–9.

    Article  CAS  PubMed  Google Scholar 

  69. Orozco-Fuentes S, Neganova I, Wadkin LE, Baggaley AW, Barrio RA, Lako M, et al. Quantification of the morphological characteristics of hESC colonies. Sci Rep. 2019;9(1):17569.

    Article  PubMed  PubMed Central  Google Scholar 

  70. Abyzov A, Mariani J, Palejev D, Zhang Y, Haney MS, Tomasini L, et al. Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells. Nature. 2012;492(7429):438–42.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Mayshar Y, Ben-David U, Lavon N, Biancotti J-C, Yakir B, Clark AT, et al. Identification and classification of chromosomal aberrations in human induced pluripotent stem cells. Cell Stem Cell. 2010;7(4):521–31.

    Article  CAS  PubMed  Google Scholar 

  72. Yang S, Lin G, Tan YQ, Zhou D, Deng LY, Cheng DH, et al. Tumor progression of culture-adapted human embryonic stem cells during long-term culture. Genes Chromosom Cancer. 2008;47(8):665–79.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors would like to thank the Tissue Engineering Research Group at the Department of Clinical Dentistry, University of Bergen, and the following funding agencies for supporting this project: The Research Council of Norway, Trond Mohn Foundation, The Western Norway Regional Health Authority and Olav Thon Foundation.

Funding

Open access funding provided by University of Bergen. This work was supported by the Research Council of Norway (Grant No. 273551) and through its Centers of Excellence funding scheme (Grant No. 22325, DEC), by Trond Mohn Foundation (Grant No. BFS2018TMT10), by the Western Norway Regional Health Authority (Helse Vest Grant No. 912260/2019, DEC) and by Olav Thon Foundation.

Author information

Author notes

  1. Kamal Mustafa and Helge Ræder contributed equally to this work and share last authorship.

Authors and Affiliations

  1. Department of Clinical Dentistry, Centre for Translational Oral Research (TOR), University of Bergen, 5009, Bergen, Norway

    Hassan R. W. Ali, Salwa Suliman, Tarig Al-Hadi Osman & Kamal Mustafa

  2. Department of Clinical Medicine, University of Bergen, Bergen, Norway

    Manuel Carrasco & Daniela-Elena Costea

  3. Centre for Cancer Biomarkers, University of Bergen, Bergen, Norway

    Manuel Carrasco & Daniela-Elena Costea

  4. Department of Medical Genetics, Haukeland University Hospital, Bergen, Norway

    Ove Bruland

  5. Gade Laboratory for Pathology, Haukeland University Hospital, Bergen, Norway

    Daniela-Elena Costea

  6. Department of Clinical Science, University of Bergen, Bergen, Norway

    Helge Ræder

  7. Department of Pediatrics, Haukeland University Hospital, Bergen, Norway

    Helge Ræder

Authors
  1. Hassan R. W. Ali
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Salwa Suliman
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tarig Al-Hadi Osman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Manuel Carrasco
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ove Bruland
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Daniela-Elena Costea
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Helge Ræder
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Kamal Mustafa
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

HRWA contributed to the design of the study, performed the experiments, analyzed and interpreted the data, and drafted the manuscript. SS contributed to the design of the study and data interpretation. DEC contributed to the design of the study and data interpretation. HR contributed to the design of the study and data interpretation. TAO contributed to data interpretation. MC contributed to the generation of the iPS. OB performed the CNV analysis. KM contributed to the design of the study and data interpretation. The authors read and approved the final manuscript.

Corresponding authors

Correspondence to Helge Ræder or Kamal Mustafa.

Ethics declarations

Ethics approval and consent to participate

Ethical approval for the collection and use of the human tissue samples was granted by the Ethical Committee for Medical and Health Related Research in West Norway (Approval number and date of approval: REK 80005, 14.06.2021). Tissue samples were collected from two healthy voluntary donors after acquiring informed consent. The title of the approved project is as follows: “Dual targeted strategy for cancer therapy and bone regeneration by use of nanodiamond loaded mesenchymal stem cells derived from adult fibroblasts.”

Consent for publication

Not applicable.

Competing interests

The authors confirm and declare that there are no known conflicts of interest associated with this publication.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1.

Overview of primers used for gene expression analysis

Additional file 2.

CNV analysis data

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ali, H.R.W., Suliman, S., Osman, T.AH. et al. Xeno-free generation of human induced pluripotent stem cells from donor-matched fibroblasts isolated from dermal and oral tissues. Stem Cell Res Ther 14, 199 (2023). https://doi.org/10.1186/s13287-023-03403-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13287-023-03403-7

Keywords

Stem Cell Research & Therapy

ISSN: 1757-6512