Rat Embryonic Fibroblasts Improve Reprogramming of Human Keratinocytes into Induced Pluripotent Stem Cells

Abstract

Patient-specific human induced pluripotent stem (hiPS) cells not only provide a promising tool for cellular disease models in general, but also open up the opportunity to establish cell-type-specific systems for personalized medicine. One of the crucial prerequisites for these strategies, however, is a fast and efficient reprogramming strategy from easy accessible somatic cell populations. Keratinocytes from plucked human hair had been introduced as a superior cell source for reprogramming purposes compared with the widely used skin fibroblasts. The starting cell population is, however, limited and thereby further optimization in terms of time, efficiency, and quality is inevitable. Here we show that rat embryonic fibroblasts (REFs) should replace mouse embryonic fibroblasts as feeder cells in the reprogramming process. REFs enable a significantly more efficient reprogramming procedure as shown by colony number and total amount of SSEA4-positive cells. We successfully produced keratinocyte-derived hiPS (k-hiPS) cells from various donors. The arising k-hiPS cells display the hallmarks of pluripotency such as expression of stem cell markers and differentiation into all 3 germ layers. The increased reprogramming efficiency using REFs as a feeder layer occurred independent of the proliferation rate in the parental keratinocytes and acts, at least in part, in a non-cell autonomous way by secreting factors known to facilitate pluripotency such as Tgfb1, Inhba and Grem1. Hence, we provide an easy to use and highly efficient reprogramming system that could be very useful for a broad application to generate human iPS cells.

Introduction

E ctopic expression of OCT4 and SOX2 in combination with both KLF4 and C-MYC [1], KLF4 alone [2], LIN28 and NANOG [3], or ESRRB [4] is sufficient to reprogram somatic cells [1,5,6], including keratinocytes derived from plucked human hair [7] to a pluripotent stem cell state. These human induced pluripotent stem (hiPS) cells have the potential to generate all tissues of the human organism and therefore provide critical access to patient-derived biomaterial in vitro [8,9]. Additionally, transplantation of autologous cells is emerging as a potential treatment strategy for numerous diseases. In addition, hiPS cells bypass the problem of immunotolerance and thereby can serve as an excellent source for tissue therapy and disease modeling.

Nevertheless, the generation of iPS cells is limited due to the low reprogramming efficiency [1] (<0.5%) and long duration [10,11] needed for reprogramming lasting several weeks in contrast to nuclear transfer or cell fusion [6]. Reprogramming efficiencies were tried to be improved by either co-expression of additional factors [10] that enhance reprogramming, for example, by chromatin modulation [12], application of chemical compounds [13], or the use of synthetic modified mRNA [14]. Particularly for the generation of patient-derived hiPS cells, a high reprogramming efficiency in a reasonable time schedule is required. Therefore, the generation of new reprogramming protocols and strategies dealing with these requirements is crucial.

hiPS cells can be generated and cultivated on mitotically inactivated feeder cells such as mouse embryonic fibroblasts (MEFs) [5] or human foreskin fibroblasts (HFFs) [15]. Feeder cells favor the process of reprogramming and the survival of the emerging stem cells via the supply of cell-to-cell contacts and the secretion of growth factors [16 –18]. Among these factors, Tgfb1, Inhba, Grem1, and Bmp4 have been reported as potential key candidates [19]. It has already been shown that these molecules are differentially secreted by MEFs after FGF2 stimulus and thereby facilitate human embryonic stem (ES) cell self-renewal [19]. This mechanism might also support the reprogramming process of somatic cells. Therefore, the type and quality of the feeder cells should have great impact on the efficiency of the reprogramming process [19]. Although human feeder cells, such as HFFs circumvent potential xeno-contaminations (eg, murine viruses and other xenobiotics), the reprogramming on HFFs remains rather inefficient and MEFs are currently the most widely used feeder cell for cellular reprogramming and hiPS cell culture [19].

We thus show that rat embryonic fibroblasts (REFs) can serve as a superior feeder cell layer compared with MEFs during reprogramming of human keratinocytes and HFFs. Arising iPS cell colonies were higher in absolute number when REFs were used instead of MEFs as feeder cell layers. This observation was further supported by an increase in absolute numbers of SSEA4 or NANOG-positive cells in REF-based reprogramming cultures. REF-based hiPS cells showed all major hallmarks of pluripotency. In addition, we found that potential candidate genes, described to be favoring stem cell survival [19], were upregulated (Tgfb1, Inhba, and Grem1) or downregulated (Bmp4) in REFs compared with MEFs, thereby pointing toward a partly non-cell autonomous mechanism. Together, we provide a very efficient reprogramming protocol for human keratinocytes, which we believe is useful for a broad application to generate human iPS cells in a large-scale format.

Materials and Methods

Keratinocyte cultures from plucked human hair

Outgrowth of keratinocytes from plucked human hair was induced essentially as previously described [7]. We used hair from 7 healthy volunteers (age between 24 to 45 and both male and female gender). Keratinocytes were consecutively cultured on 20 μg/mL collagen IV (Sigma-Aldrich, St. Louis, MO; www.sigmaaldrich.com)-coated dishes in EpiLife medium with HKGS supplement (both Invitrogen, Carlsbad, CA; www.invitrogen.com) for up to 2 passages. The use of human material in this study is approved by the ethics committee of the Ulm University (No. 0148/2009) and in compliance with the guidelines of the Federal Government of Germany and the Declaration of Helsinki concerning Ethical Principles for Medical Research Involving Human Subjects.

MEF and REF culture, HFFs, and FGF2 producing immortalized HFFs

CD-1 MEFs from embryonic day E12.5 or E14.5 (Stemcell Technologies, Vancouver, CA; www.stemcell.com) were cultivated according to manufacturer's protocol or isolated from CD-1 or C57BL/6J (E12.5 and E13.5) mice by standard protocols [1]. CF-1 MEFs from embryonic day 13.5 (Applied StemCell, Menlo Park, CA; www.appliedstemcell.com) were cultivated equally to the CD-1 MEFs. REFs were isolated from day E14 Sprague Dawley rat embryos as described earlier for mice [1] and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum (FBS), 2 mM GlutaMAX, 100 μM nonessential amino acids, and 1% antibiotic–antimycotic (all Invitrogen). Cells were passaged by 0.125% trypsin digestion when reaching confluence for up to 5 passages. Normal HFFs (System Biosciences, Mountain View, CA; www.systembio.com) were cultured according to manufacturer's protocol. FGF2 producing immortalized HFFs (kind gift of A.Feki, Geneva, Switzerland) were cultured as described [15]. We used 10 independent REF preparations for the reprogramming experiments. All animal experiments were performed in compliance with the guidelines for the welfare of experimental animals issued by the Federal Government of Germany, the National Institutes of Health, and the Max Planck Society (Nr. O.103).

Human iPS cell culture

hiPS cells were kept in an incubator with 5% O₂ on feeder cells in Knockout DMEM supplemented with 20% Knockout Serum Replacement (Invitrogen), 2 mM GlutaMAX, 100 μM nonessential amino acids, 1% antibiotic–antimycotic, 100 μM β-mercaptoethanol, 50 μg/mL ascorbic acid, and 10 ng/mL FGF-2 and passaged mechanically or by scratching after collagenase-IV treatment. For later passages hiPS cells were transferred to Matrigel-coated dishes (BD Biosciences, Franklin Lakes, NJ; www.bd.com), kept in mTeSR1 medium (Stemcell Technologies), and passaged according to the manufacturer's manual.

For cultivation in conditioned medium, irradiated CF-1 MEFs or REFs were kept in the medium described above but with 5 ng/mL FGF-2. The medium was collected for 5 days, sterile filtered, and stored at 4°C. One day after seeding cells on Matrigel in mTeSR1 the medium was changed to a 50:50 mixture of mTeSR1 and conditioned medium. From the following day on, the cells were kept in the conditioned medium supplemented with an additional 5 ng/mL FGF-2. Passaging was performed mechanically.

Lentivirus generation

Lentivirus containing a polycistronic expression cassette encoding for Oct4, Sox2, Klf4, and c-Myc [20] was produced in 70% confluent 10 cm dishes with Lenti-X 293T cells (Clontech, Mountain View, CA; www.clontech.com) by cotransfection of the polycistronic vector (8 μg), the pMD2 vector (2 μg), and the psPAX2 (5.5 μg) vector (both Addgene, Cambridge, MA; www.addgene.org) using 100 μL of the PolyFect transfection reagent (Qiagen, Hilden, Germany; www.qiagen.com). Viral supernatant was collected at 48 and 96 h after transfection, concentrated using the Lenti-X Concentrator Kit (Clontech), resuspended in EpiLife medium, and stored in aliquots at −80 °C.

Reprogramming of keratinocytes

For infection, up to 3×10⁵ keratinocytes per well of a 6-well plate were infected with 5×10⁷ proviral genome copies in EpiLife medium containing 8 μg/mL Polybrene (Sigma-Aldrich) at 2 sequent days. Another 24 h later cells were detached using TrypLE Express (Invitrogen) and distributed onto 3 wells of a 6-well plate with already attached irradiated feeder cells (3×10⁵ cells per well irradiated with 30 Gy) in hiPS cell medium. Further on the cells were cultured in a 5% O₂ incubator. The medium was changed daily until arising colonies were big enough for mechanical passaging at about 2–3 weeks after transduction. Colonies displaying a clear stem cell morphology were picked and transferred onto irradiated MEFs or Matrigel-coated plates for further passage.

Germ layer differentiation and teratoma formation

Several hiPS cell colonies were detached by dispase treatment and kept in a ultra-low attachment suspension culture flask (Corning, Corning, NY; www.corning.com) for 10 days in Knockout DMEM supplemented with 20% Knockout Serum Replacement, 2 mM GlutaMAX, 100 μM nonessential amino acids, 1% antibiotic–antimycotic, and 100 μM β-mercaptoethanol with daily medium change. For adherent cultures the forming embryoid bodies (EBs) were seeded on Matrigel-coated plates and kept in culture for up to 14 days. For the teratoma, formation assay human iPS cells (1–2×10⁶ cells/mouse) were injected subcutaneously into the dorsal flank of severe combined immunodeficient (SCID) mice. Six weeks after injection, teratomas that had formed, were fixed in 4% paraformaldehyde (PFA) overnight and embedded in paraffin. Sections were stained with hematoxylin and eosin dyes using standard protocols.

Immunocytochemistry and alkaline phosphatase staining

Immunofluorescence staining was performed according to [21,22] and the manufacturer's protocol using the StemLite Pluripotency Kit (Cell Signaling, Danvers, MA; www.cellsignal.com) and Alexa secondary antibodies (Invitrogen). For germ layer-specific detection we used the chicken anti Tubulin β-III antibody (TUBB3) (1:1000; Millipore, Billerica, MA; www.millipore.com), mouse anti Actinin antibody clone EA-53 (1:150; Sigma-Aldrich), goat anti AFP (C-19) antibody (1:100; Santa Cruz, Santa Cruz, CA; www.scbt.com), and Alexa secondary antibodies. Cells were mounted with the ProLong Gold Antifade Reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen).

For alkaline phosphatase (AP) staining, cells were rinsed in phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 2 min. Cells were then stained in a buffer containing 0.3 mg/mL Nitro Blue Tetrazolium and 0.175 mg/mL 5-Bromo-4-chloro-3-indolyl phosphate p-toluidine salt for 15–30 min in the dark until a clear blue staining was visible. The staining reaction was stopped by washing with PBS.

Western blots

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blots were carried out according to standard procedures [23,24]. The following antibodies were used: TGFB1 polyclonal rabbit antibody (1:400, PAB12738; Abnova, Taipei, Taiwan; www.abnova.com), INHBA polyclonal rabbit antibody (1:250, PAB5409; Abnova), GREM1 polyclonal rabbit antibody (1:400, PAB14845; Abnova), BMP4 polyclonal rabbit antibody (1:1000, PAB9482; Abnova), beta-ACTIN monoclonal mouse antibody (1:5000, Clone AC-74; Sigma-Aldrich), and polyclonal swine anti-rabbit HRP-conjugated antibody (Dako, Glostrup, Denmark; www.dako.com). Irradiated MEFs and REFs were kept in hiPSC medium for 3 days before lysis. Protein concentrations were measured after amido black staining to ensure equal loading.

Quantitative one-step real-time reverse transcription-polymerase chain reaction

Quantitative one step real-time reverse transcription-polymerase chain reaction (qRT-PCR) was carried out according to [8,25] using the Rotor-Gene Q System (Qiagen, Hilden, Germany, www.qiagen.com), and amplification was monitored and analyzed by measuring the binding of the fluorescence dye SYBR Green I to the minor groove of double-stranded DNA. One microliter of total RNA, extracted with the RNeasy Kit (Qiagen), was reversely transcribed and subsequently amplified using QuantiFast SYBR Green RT-PCR Master mix (Qiagen). About 0.5 μmol of both sense and antisense primers was added. To optimize the real time-PCR conditions, optimal MgCl₂ concentration and annealing temperature were determined. Each RNA preparation was tested for genomic DNA contamination in a Rotor-Gene Q RT-PCR by replacing reverse transcriptase with water. Tenfold dilutions of total RNA were used as an external standard curve. PCR efficiency results from the slope of these standard curves. Internal standards (housekeeping gene) and samples were simultaneously amplified. RT-PCR conditions started with 10 min of reverse transcription at 50°C, followed by activation of Taq-polymerase at 95°C for 5 min. Forty-five cycles with denaturation at 94°C for 10 s and 60°C for 30 s amplified all described products with a PCR efficiency close to 3.3 (exact doubling of PCR-product). To verify the specificity of the PCR amplification products, melting curve analysis was performed using the following thermal cycling profile: 95°C for 0 s, 65°C for 15 s, and ramping to 95°C with stepwise signal acquisition. The concentration of each transcript was calculated by reference to the respective standard curve. Relative gene expression was expressed as a ratio of target gene concentration to housekeeping gene hydroxymethylbilane synthase concentration. Quantitect primer assays (Qiagen) were used in all experiments except for the detection of the polycistronic cassette. For this, the primer pair 5′-caggctgtggcaaaacctat-3′ and 5′-ccctaggaatgctcgtcaag-3′ was used.

Gene expression microarray

Gene expression microarray was carried out using the Agilent Whole Human Genome Microarray Kit (4x44k microarray kit G4112F; Agilent Technologies, Santa Clara, CA; www.home.agilent.com). Cy3-CTP-labeled cRNA was produced from 500 ng of total RNA using the Agilent Low RNA Input Liner Amplification Kit. After labeling and cRNA purification, cRNA was quantified using the NanoDrop ND-1000 UV-VIS Spectrophotometer. Cy3-labeled cRNA (1.65 μg) was hybridized per individual array for 17 h at 65°C (10 r.p.m.). After hybridization, arrays were washed consecutively with Agilent Gene Expression Wash Buffers 1 and 2, and acetonitrile for 1 min each. Slides were scanned immediately after washing in the Agilent Scanner using Scan Control 7.0 software. The scan resolution was 5 μm. Scan data were extracted with Feature Extraction 9.1 software. Gene expression levels were background adjusted and quantile normalized. Hierarchical clustering with Pearson correlation coefficient and average linkage was performed by hclust function in R package stats and represented by dendrogram or heatmap. Differential expression was analyzed using Student's t-tests. Transcripts with a fold change >2 and P value <0.05 between 2 cell types were considered as being differentially expressed. Raw P values were adjusted by Bonferroni–Holm procedure [26]. Data sets were compared with available sets from GEO (www.ncbi.nlm.nih.gov/geo/).

Flow cytometry

Cells were detached by trypsin digestion and washed in buffer (2% FBS, 2 mM EDTA, and 20 mM Glucose in PBS). For intracellular staining, cells were fixed for 20 min in 2% PFA on ice and permeabilized with 0.5% saponin for 20 min on ice. Cells were resuspended in buffer containing primary antibody (SSEA4 antibody, 1:100, Cell Signaling, BrdU antibody, 1:200; BD Biosciences). After 30 min of shaking at 4°C the cells were washed twice, resuspended in buffer containing Alexa-647 secondary antibody (Invitrogen), and kept for 30 min at 4°C in the dark while shaking. Afterward the cells were again washed twice, resuspended in buffer, and filtered through a 100 μm filter. DAPI (5 μg/mL) was added just before analysis if unpermeabilized cells were used. Measurements were performed in an LSRII flow cytometer using the FACSDiva software (both BD Biosciences).

Karyotyping

Chromosome preparation of trypsinized keratinocyte-derived hiPS (k-hiPS) cells of passages 10–20 was performed according to standard procedures, and karyotyping was done after GTG-banding. A total of 38 metaphases were scored.

Bisulfite sequencing

Genomic DNA was isolated from different k-hiPSC lines using the QIAamp DNA Mini Kit (Qiagen). Three hundred nanograms was used as input for bisulfite conversion (EpiTect Bisulfite Kit; Qiagen). Fifty nanograms of converted DNA was used as a template for conventional nested PCRs amplifying regions of the OCT4 and NANOG promoters using primers described earlier [27]. Purified PCR products were TA-cloned into pCR2.1-TOPO (Invitrogen), sequenced, and analyzed as previously described [27].

Statistical analysis

Reprogramming efficiency was calculated by dividing the average count of AP-positive colonies per well by the initial number of cells plated, scaled to the fraction of cells replated in each well. All quantitative data are expressed as mean±standard error of the mean. Statistical comparisons were made by Dunnett's t-test or Student's t-test and considered significant if P values were <0.05 (*) and <0.01 (**). Calculations of significance were done with GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA; www.graphpad.com).

Results

REFs as feeder cells during reprogramming of human keratinocytes

REFs were isolated and cultured similar to MEFs and showed a similar morphology (Fig. 1A, B) and growth behavior. To determine optimal conditions for the reprogramming on MEFs as a feeder layer, we initially tested MEFs of different origin (Supplementary Fig. S1; Supplementary Data are available online at www.liebertonline.com/scd), embryonic age, and mouse strain. To narrow down side effects in our culture system, throughout this study we used commercially produced MEFs (E14.5, CD-1) to compare the REF-based system with standard MEF strains. Plucked human hair has been successfully used to obtain keratinocytes for reprogramming into k-hiPS cells [7]. This technique needed first to be reproduced and optimized especially since there are very few further reports applying this promising technique (Fig. 1C) in a routine manner [28]. Human head hair was plucked from healthy individuals and cultivated to obtain keratinocyte outgrowth (Fig. 1D). Outgrowing keratinocytes could be passaged for several passages (Fig. 1E). Next, keratinocytes were infected with a polycistronic lentivirus encoding all 4 reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) [20] and reseeded on inactivated MEF or REFs. The medium was changed from keratinocyte conditions to hiPS cell conditions. Seven to 8 days after infection of the keratinocytes, early k-hiPS cell colonies started to appear under the ascribed culture conditions (Fig. 1F). The colonies had a tightly packed, flattened morphology resembling human ES cell colonies. The reprogramming of keratinocytes on REFs led to numerous k-hiPS cell colonies with typical compact morphology and prominent nucleoli, which were picked and passaged for further analysis in a feeder free system (Fig. 1G).

FIG. 1.

Human keratinocytes can be reprogrammed on MEFs and REFs using the same protocol. (A) Differential interference contrast (DIC) microscopy images of irradiated MEFs and (B) REFs at a density of 3.1×10⁴ cells per cm² before addition of infected keratinocytes. (C) Schematic procedures of keratinocyte reprogramming. Plucked human hair was used as a source for keratinocytes. After the initial outgrowth, the keratinocytes were infected with a lentivirus carrying the reprogramming factors Oct4, Sox2, Klf4, and c-Myc. Two days later they were transferred onto a feeder layer of MEFs or REFs until the arising colonies were mechanically passaged. (D) DIC microscopy images of keratinocyte outgrowth from a plucked hair. (E) Confluent keratinocytes at time point of lentiviral infection. (F) Arising k-hiPS cell colonies on REFs 10 days after infection and (G) a k-hiPS cell colony growing on Matrigel in mTeSR1 medium. All scale bars, 100 μm. MEF, mouse embryonic fibroblast; REF, rat embryonic fibroblast; hiPS, human induced pluripotent stem; k-hiPS, keratinocyte-derived hiPS.

REFs as a feeder layer improve reprogramming of keratinocytes

To directly compare the reprogramming efficiency of both feeder types, infected keratinocytes were seeded at identical cell numbers on either inactivated MEFs or REFs of the same passage. A few hours later, viable cells attached on both feeder cell types in comparable numbers. Seven to 8 days after infection, keratinocytes displayed strong alterations in cell morphology. They started to form tightly packed colonies that flattened during ongoing culture. Within 14–21 days colonies emerged that were big enough for mechanical passaging (Fig. 2A). On day 14, cultures were stained with AP (Fig. 2B). The total number of AP-positive colonies was about 3-fold higher on REFs compared with MEFs, leading to a reprogramming efficacy of ∼2.8% on REF feeder layers (Fig. 2C). To apply a more stringent assay for cellular reprogramming, namely, flow cytometry, we aimed to determine the amount of SSEA4-positive cells using either REFs or MEFs as feeder cell layer (Fig. 2D). On day 14 after infection the number of SSEA4-positive cells was about 2-fold higher in REF feeder layer-based cultures (Fig. 2E). There were no relevant differences in reprogramming performance between 4 REF preparations (Fig. 2C, E). In addition, REF-based reprogramming efficacies were independent of passage numbers (Supplementary Fig. S2). Reprogramming on REFs was superior to MEFs in all keratinocyte samples generated from 7 donors. In contrast, the use of FGF-2 producing immortalized HFFs that have been shown to be useful as feeder cells for higher hiPS cell passages [15] led to poor reprogramming efficiencies (below 0.001%, not shown).

FIG. 2.

REFs have a significantly higher suitability as feeder cells for keratinocyte reprogramming compared with MEFs. (A) Emerging colonies on REFs look comparable to those on MEFs. Scale bars, 100 μm. (B) Two weeks after seeding the same amount of keratinocytes on different feeder cells, an AP staining was performed and colony numbers were counted. (C) The number of emerging AP-positive k-hiPS cell colonies is approximately 3-fold higher on REFs compared with MEFs (n=4 independent experiments). Control reprogramming efficiencies on MEF cultures were 0.8%±0.7%. (D) Two weeks after seeding infected keratinocytes on feeder cells, emerging k-hiPS cells were analyzed by flow cytometry. SSEA4-positive k-hiPS cells are shown as a distinct population in flow cytometry plots. The populations have identical fluorescence and scattering characteristics on MEFs and REFs. (E) The ratio of SSEA4-positive k-hiPS cells to total cell counts is about 2-fold higher on REFs compared with MEFs (n=3). Quantitative results are mean values±SEM from at least 3 independent experiments. AP, alkaline phosphatase; SEM, standard error of the mean. *, P<0.05; **, P<0.01.

REF-based k-hiPS cells show all major pluripotency characteristics

Given the strongly enhanced reprogramming potency of REF feeder layers, we analyzed the quality of the generated k-hiPS cell clones according to established protocols (see Materials and Methods section). We generated numerous k-hiPS cell clones on REFs from different donors, which all harbored typical morphology and growth rates of human ES cells. All established clones stained positive for the stem cell markers OCT4, NANOG, SOX2, SSEA4, TRA-1-60, and TRA-1-81 in immunocytochemical analysis (Fig. 3A). In addition, all tested clones showed high endogenous expression levels for OCT4, SOX2, and NANOG, whereas KLF4 showed lower expression levels (also described in ref. [7]) when compared with keratinocytes (Fig. 3B). Global gene expression of k-hiPSC clustered close to hES cells and hiPS cells established elsewhere (Fig. 3C, D). Scatter plots analysis showed a high similarity between k-hiPSC and human ES cells, but not between k-hiPSC and keratinocytes (Fig. 3E). Thus, k-hiPSC are close to human embryonic stem cells at the global transcription level. Chromosome analysis of k-hiPSC showed that the majority (88%) has a normal chromosome set (Fig. 3F). As expected in fully reprogrammed hiPS cells, the promoter regions of the endogenous OCT4 and NANOG genes were fully demethylated (Fig. 3G) and expression of viral vector-encoded genes was silenced in our k-hiPS cell colonies (Fig. 3H).

FIG. 3.

Stem cells produced on REFs as a feeder layer show major pluripotency characteristics. (A) Immunofluorescence staining of the nuclear factors OCT4, NANOG, and SOX2 and surface markers SSEA4, TRA-1-60, and TRA-1-81. All scale bars are 100 μm. (B) Expression levels of endogenous OCT4, SOX2, KLF4, and NANOG were compared between keratinocytes (Ker) and k-hiPS cells. Exemplary results show an upregulation of OCT4, SOX2, and NANOG and a downregulation of KLF4 in k-hiPS cells. (C) Heatmap of human transcriptome microarray comparing keratinocytes, k-hiPSC, and hESC. The differently regulated genes between keratinocytes and k-hiPSC are selected. Downregulation is shown in blue and upregulation is shown in red. (D) Dendrogram analysis of k-hiPSC gene expression profile shows a high similarity between k-hiPSC and already published hiPSC and hESC data (GEO accession GSE21792 and GSE7902). (E) Scatter plot of k-hiPSC gene expression compared with keratinocytes or hESC. Typical pluripotency markers are expressed similarly in k-hiPSC and hESC while differing in keratinocytes. (F) Exemplary karyogram of a k-hiPS cell. Karyotype analysis of 26 cells showed 23 normal karyotypes (88%). (G) Bisulfite sequencing of the OCT4 and NANOG promoter in k-hiPSC shows almost no methylation permitting transcriptional activity of these genes. (H) Silencing of the transduced viral cassette was confirmed with qRT-PCR using primers lying inside the cassette, specifically amplifying the exogenous factors. P0 represents reprogrammed keratinocytes 2 weeks after viral transduction; P10 represents k-hiPS cells at 10 passages after first mechanical picking. Shown is 1 representative k-hiPS cell line. hESC, human embryonic stem cell.

REF-based k-hiPS cells differentiate into all 3 germ layers in vitro and in vivo

A hallmark feature of pluripotent stem cells is their potency to differentiate into all 3 germ layers. To confirm this differentiation pattern on protein level, marker proteins were analyzed by immunostaining. REF-based, k-hiPS cell-derived EBs were capable to form mesoderm and subsequently striated muscle cells as shown by α-actinin staining. Neuronal differentiation potential was confirmed by the neuronal marker β3-tubulin (TUBB3). In addition, α-feto-protein staining (AFP) depicted multi-nucleated cells pointing to endodermal, early hepatic differentiation of the cultures, representing the endoderm (Fig. 4A). mRNA expression analysis via quantitative RT-PCR (qRT-PCR) showed upregulation of marker genes for neural/ectoderm differentiation (PAX6, TUBB3), endodermal/hepatic differentiation (AFP, FOXA2), and mesoderm/cardiac differentiation (MYH6, T) (Fig. 4B) during EB differentiation.

FIG. 4.

The stem cell colonies established on REFs are able to differentiate into cells of all 3 germ layers in vitro and in vivo. hiPS cell colonies were detached and kept in suspension for 10 days to form embryoid bodies. These were seeded on Matrigel-coated slides and stained after additional 14 days. (A) Immunofluorescence stainings show cells positive for ectodermal (TUBB3: Tubulin beta-III), mesodermal (α-Actinin), and endodermal (AFP: Alpha fetoprotein) marker proteins. Scale bars, 20 μm. (B) Upregulation of several marker genes was measured by quantitative one-step real-time reverse transcription-polymerase chain reaction (qRT-PCR). The endodermal markers AFP and FOXA2, the mesodermal markers MYH6 and T, as well as the ectodermal markers TUBB3 and PAX6 are upregulated compared with undifferentiated k-hiPS cells (n=3). (C) Teratomas formed by k-hiPS cells after injection into SCID mice contain ectodermal (neuroectoderm), mesodermal (cartilage), and endodermal (respiratory epithelium) tissue showing that k-hiPS cells can differentiate into all 3 germ layers in vivo.

The in vivo potential of REF-based k-hiPS cells was assessed by teratoma formation in SCID mice, which contained tissues originating from all 3 germ layers (Fig. 4C).

REFs favor reprogramming and stem cell self-renewal non-cell autonomously

Proliferation rates have been shown to influence reprogramming efficiencies [10,29]. Although under debate, it has been proposed that proliferation rate is the major factor inducing stochastic events that lead to reprogramming [10,29,30]. To analyze whether differences in keratinocyte or k-hiPS cell proliferation due to different feeder layers could explain increased reprogramming efficiencies, proliferation rates were analyzed at early time points after virus infection of keratinocytes. BrdU labeling for 6 h revealed no differences in proliferation rates when being reprogrammed on MEFs or REFs (Fig. 5A). Another explanation for increased keratinocyte reprogramming on REFs could be a non-cell autonomous action such as secretion of specific factors enhancing hiPS formation. To that end culture medium was conditioned on REFs for 24 h and then used for reprogramming keratinocytes on MEFs. REF-conditioned medium enhanced the reprogramming on MEFs and led to an intermediate reprogramming efficiency between those on MEFs and REFs with fresh medium (Fig. 5B). In case of a non-cell autonomous action, REFs should be also able to enhance reprogramming of other cell types than keratinocytes. In this respect, we compared hiPS formation from HFF on MEF or REF feeder layers. In fact, REF-facilitated reprogramming was also superior when HFFs were used as parental cell type (Fig. 5C). Finally, we assessed the capacity of REFs to facilitate stem cell culture in the undifferentiated stage. Both REFs themselves and their conditioned medium enabled k-hiPS cells to be cultured for several passages without loss of pluripotency as assessed by morphology and OCT4 and NANOG protein expression (Fig. 5D, E; Supplementary Figs. S3 and S4).

FIG. 5.

REFs favor reprogramming but not proliferation in a non-cell autonomous way. (A) The proliferation rate of k-hiPS cells at 5 days after keratinocyte infection is equal on MEFs and REFs as shown by 1 μg/mL BrdU labeling for 6 h and subsequent flow cytometry. (B) Addition of REF conditioned medium to reprogramming keratinocytes on MEFs leads to a higher number of arising k-hiPSC colonies compared with reprogramming on MEFs alone. Medium with 5 ng/mL FGF-2 was conditioned on irradiated REFs for 24 h and additional 5 ng/mL FGF-2 was added before use. Colony numbers were evaluated by AP stainings. (C) HFFs were infected according to the keratinocyte experiments and seeded onto MEF or REF feeder layers. Two weeks after infection the ratio of NANOG positive cells was measured by flow cytometry. NANOG was chosen since SSEA4 antibodies produced high background in unreprogrammed HFFs. (D) REFs can sustain k-hiPS cell growth over several passages. After 5 passages on REFs or MEFs k-hiPSC colonies were detached, seeded on Matrigel in mTeSR1 medium overnight, and stained the next day. Colonies are positive for the pluripotency markers OCT4 and NANOG in immunofluorescence. All scale bars are 100 μm. (E) DIC images of k-hiPSC colonies in REF conditioned medium show comparable morphology to colonies in CF-1 MEF conditioned medium after 5 passages. Scale bars are 100 μm. *, P<0.05.

REFs express distinct factors facilitating reprogramming and self-renewal

Having established several lines of evidence pointing toward a non-cell autonomous mechanism of REF-facilitated reprogramming, we aimed to identify at least some of these factors being present in REFs when compared with MEFs. It has been shown that TGFbeta signaling cascade is crucial for human pluripotent stem cell self-renewal, whereas BMP-signaling needs to be repressed to prevent differentiation [19]. qRT-PCR analysis of REFs growing in hiPS cell medium showed a significantly higher expression of key members of the TGFbeta pathway, Tgfb1, Inhba, and Grem1 compared with MEFs (Fig. 6A–C), whereas Bmp4, which favors differentiation, is downregulated (Fig. 6D). These observations could also be confirmed on protein level via western blot analysis of Tgfb1, Inhba, Grem1, and Bmp4 (Fig. 6E).

FIG. 6.

Analyses of potential candidate genes mediating the improvement of reprogramming by REF feeder layers by qRT-PCR and western blot. qRT-PCR show higher mRNA levels of the candidate factors for high reprogramming efficiency (A) Tgfb1, (B) Inhba, and (C) Grem1 in REFs compared with MEFs, whereas (D) Bmp4 mRNA levels were nonsignificantly lower in REFs compared with MEFs. Results are mean values±SEM from at least 3 independent experiments. *P<0.05 when compared with MEFs. (E) Western blot results confirm an upregulation of protein levels of Tgfb1, Inhba, and Grem1 and a downregulation of Bmp4 in REFs compared with MEFs. Equal loading was confirmed by Actin detection and by amido black protein staining and subsequent concentration measurement.

Discussion

One of the most promising applications for hiPS cells is the acquisition of patient-specific stem cells and their use as a patient-specific disease model and readout system for pathophysiological and treatment research [31 –33]. Moreover, this novel technology provides individual cell systems for personalized medicine either by autologous cell replacement approaches or by person- and cell type-specific testing. The optimal tissue and cell type for reprogramming strategies is, however, still a matter of debate. Moreover, epigenetic memory preventing certain iPS cell lines to differentiate into a particular lineage has to be taken into account [34,35]. Most laboratories are currently using skin biopsy-derived fibroblasts that are highly proliferative and can be passaged over time. Nevertheless, reprogramming efficiencies remain low and the process is rather time-consuming and costly. Additionally, skin biopsies are more invasive compared with sampling plucked hair. Indeed, keratinocyte reprogramming has been reported to be 100-fold more efficient with reprogramming efficiencies of around 1% and 2-fold faster than fibroblast reprogramming despite using MEFs instead of our REF-based reprogramming system [7]. One of the reasons for the high reprogramming efficiencies in keratinocytes may be associated with transcriptional and epigenetic states favorable to reprogramming due to high endogenous levels of KLF4 and C-MYC in keratinocytes [7]. Similarly, a high reprogramming efficiency has been reported for neural stem cells, even when using only KLF4 and OCT4 as reprogramming factors [36]. Nevertheless, neural stem cells are doubtless not a broadly available cell source allowing the generation of patient-specific iPS cell libraries. Another, more noninvasive method favoring reprogramming is the usage of peripheral blood [37], but purification of the desired cell type is rather costly and difficult [38]. Cells that are easy to obtain are keratinocytes from the outer root sheath of human hair. This cell type is initially highly proliferative and can be propagated for several passages. To further improve reprogramming, we combined several known enhancements of the protocol to make it even more efficient such as the use of a polycistronic reprogramming construct [20,28], the use of low oxygen atmosphere [39], and the addition of ascorbic acid [40] or valproic acid [41]. However, most of these studies used feeder cell layers as a crucial factor for the survival and support of cells during the reprogramming process with MEFs being the most commonly used feeder cell type. Still, feeder cells display various and differential disadvantages. Indeed, MEF feeder cell lines are not only differing among preparations, but their quality also depends on age and strain of mice [19,42]. We identified REFs not only as an alternate and more stable feeder cell source but rather as an enhancer of human keratinocyte reprogramming. This has been shown by a significant increase in the number of reprogrammed cells and number of arising hiPS cell colonies on REF feeder layers with an estimated reprogramming efficacy of up to 2.8%. However, it is very difficult to compare reprogramming efficiency numbers between scientific groups, since there are a variety of different reprogramming protocols in use, including different vectors, virus amounts, feeder treatment, and, finally, experiment evaluation. To check if the reprogramming favoring effect of REFs is limited to keratinocytes, we reprogrammed HFFs and found that the efficiency was enhanced by more than 50% on REFs compared with MEFs. This indicates that the enhancement is not specific for the cell type being reprogrammed but for the used feeder cell layer. These data underline recent findings indicating that the environment of the cells being reprogrammed is crucial for their fate [43]. Therefore, REFs seem to produce a more suitable environment especially for early events in reprogramming.

To narrow down side effects in our culture system, we tested commercially produced MEFs to compare the REF-based system with standard MEF strains. The REF-based system was superior to all tested MEF batches and strains. Indeed, every REF preparation was successfully suitable for efficient reprogramming of keratinocytes pointing to increased reproducibility of REFs due to being less biased by culture conditions. REFs also maintained their reprogramming enhancing abilities over several passages. Although we did not test different reprogramming methods such as plasmid or RNA transfection, it is very likely that the use of REFs also eases reprogramming in other settings. Using REFs as feeder cells we successfully reprogrammed keratinocytes from several donors. The established k-hiPS cell lines showed the expected stem cell morphology and growth behavior, expressed all common stem cell markers, exhibited a gene expression profile very similar to hiPS and hES cell lines from other labs, and showed a mostly normal karyotype. Additionally, they had a silenced reprogramming cassette, demethylated promoters of typical stem cell marker genes, and could differentiate into various cell types in vitro and in vivo, comparable to stem cells obtained on MEFs. Further effort has been invested to delineate the mechanism behind the optimized reprogramming efficiency of REFs when compared with MEFs. Although under debate, it has been proposed that proliferation rate is the major factor inducing stochastic events that lead to reprogramming [10,29,30]. Nevertheless, REF-based reprogramming does not affect proliferation rates of the cells to be reprogrammed. Moreover, there seems to be a non-cell autonomous mechanism facilitating the reprogramming of keratinocytes by secreted factors. This has been shown by conditioned medium experiments and reprogramming of different cell types on REFs. We found that several genes, linked to self-renewal of hiPS cells [19], were expressed higher in REFs compared with MEFs, namely, Tgfb1, Inhba, and Grem1. Tgfb1 and Inhba code for secreted factors from the TGFβ pathway that enhance self-renewal through activation of SMAD2/3, whereas Grem1 inhibits Bmp4 signaling. On the other side, Bmp4 was downregulated in REFs compared with MEFs. This self-renewal favoring expression profile of REFs could well be the reason for their high suitability as feeder cells in the critical stages of reprogramming but also for long-term culture of hiPS cells. Future studies are warranted to finally confirm the responsible factors and/or the respective signaling pathways, allowing the generation of efficient reprogramming protocols without the use of feeder cell layers or—at least—without the use of xenobiotic material.

In summary, we provide a feeder cell system that is superior to the commonly used cell types employed for the support of human iPS cell reprogramming in terms of reprogramming efficiency. This system was generated to further facilitate the reprogramming of human plucked hair-derived keratinocytes. This unique complementation of a non-invasive to obtain cell source that harbors an endogenously high reprogramming potential together with a superior and more robust feeder system provides a pivotal prerequisite for reliable and fast generation of patient-specific k-hiPS cells to be applied in personalized medicine.

Footnotes

Acknowledgments

A.K. is supported by a fellowship provided by the Medical Faculty of Ulm University (Bausteinprogramm, L.SBR.0011). Main part of this study was funded by the Deutsche Forschungsgemeinschaft (DFG) (A.K. SFB 518 S.L., T.M.B. SFB 497/B8 and S.L., T.M.B. BO1718/4-1). The authors would like to thank Sabine Seltenheim, Renate Zienecker, Martina Bleidißel, and Boris Burr for their technical assistance.

Author Disclosure Statement

The authors indicate no potential conflicts of interest.

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