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Footprint- and xeno-free human iPSCs derived from urine cells using extracellular matrix-based culture conditions
Biomaterials 35 (2014) 8330e8338
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Footprint- and xeno-free human iPSCs derived from urine cells using extracellular matrix-based culture conditions Kang-In Lee 1, Hyeong-Taek Kim 1, Dong-Youn Hwang* Department of Biomedical Sciences, CHA University, Seongnam, Kyeonggido 463-840, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 March 2014 Accepted 21 May 2014 Available online 30 June 2014
The efficient generation of integration- and xeno-free iPSCs is a prerequisite for their use in clinical applications. Furthermore, non-invasiveness of somatic cell acquisition for iPSC generation is another factor to consider. In this study, we established a practical, simple, and convenient method to generate integration- and xeno-free iPSCs from urine cells which can be obtained in a non-invasive manner. Our method was based on extracellular matrix-based xeno-free iPSC culture condition and episomal transfection, and worked efficiently with both urine cells and adipose-derived stromal cells (ADSCs). To obtain strictly xeno-free iPSCs, we also formulated a new xeno-free culture medium for primary urine cells. Intriguingly, urine cells displayed slower growth, and more dramatic increase in apoptosis at high passage numbers than ADSCs. However, urine cells at low passage (
Keywords: Integration-free Xeno-free Urine cells Induced pluripotent stem cells Cell therapy
1. Introduction One of the most valuable advantages of human induced pluripotent stem cells (iPSCs) is their potential for use in immunecompatible cell replacement therapy. For cell therapy purposes, the generation of footprint- and xeno-free iPSCs is one of the major concerns that remains to be addressed. In this regard, rapid progress has been made in reprogramming somatic cells without the insertion of transgenes into the chromosomes. Until now, the overexpression of reprogramming factors using EBNA1/OriP (Epstein-Barr nuclear antigen Origin of plasmid replication)-based plasmids [1e3], mRNAs [4], and Sendai viruses [5] has attracted significant attention as methods that generate footprint-free iPSCs. Another important safety-related issue is the establishment of xeno-free conditions for both the derivation and expansion of the iPSCs. Conventionally, human pluripotent stem cells (hPSCs) were cultured on mouse embryonic fibroblast feeder cells using culture media containing animal-derived components. The mouse feeder cell-dependent culture system faced potential risks, including the transmission of non-human pathogens to humans and immune
* Corresponding author. Tel.: þ82 31 8017 9411; fax: þ82 31 750 9447. E-mail address: [email protected] (D.-Y. Hwang). 1 Both authors contributed equally to this work. http://dx.doi.org/10.1016/j.biomaterials.2014.05.059 0142-9612/© 2014 Elsevier Ltd. All rights reserved.
rejection problems elicited by contamination from non-human antigens [6,7]. Therefore, an extracellular matrix (ECM)-based feeder-independent culture system is of great value to produce and culture xeno-free human iPSCs. With the initiation of a clinical trial using iPSCs for the treatment of macular degeneration, the development of method to generate xeno- and footprint-free iPSCs became more important than ever. In this regard, recent reports demonstrated successful generation of iPSCs using xeno-free media in combination with xeno-free ECMs, such as vitronectin [8], laminin [9], and pericellular matrix of decidua-derived mesenchymal stem cells [10]. Another important issue to consider for the generation of iPSCs from patients is the source of somatic cells that are used for iPSC generation. A variety of cell types, including dermal fibroblasts [11], bone marrow CD34 þcells [12], cord blood cells [13,14], peripheral blood cells [15e17], adipose-derived stromal cells [18], neural stem cells [19], and keratinocytes [20], have been used to generate iPSCs. There is a growing consensus that an ideal cell source for iPSC generation should be acquired easily and noninvasively, and the efficiency of iPSC generation should be considerably high. Recently, urine cells were proposed as a promising cell source for iPSC generation, as these cells can be obtained in a non-invasive manner [21]. In this study, we sought to establish an efficient method to generate integration- and xeno-free iPSCs by elaborately combining
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an ECM-based feeder-independent/xeno-free hPSC culture system [22] and episomal vector transfection method. Furthermore, we examined if the combined iPSC-generating system functioned for urine cells which are non-invasively obtainable cells in the human body. The generation of integration- and xeno-free iPSCs from urine cells would provide a convenient and easy-to-access platform for iPSC-based cell replacement therapies in the near future. 2. Materials and methods 2.1. The isolation and culture of urine cells and adipose-derived stromal cells (ADSCs) This study was approved by the Institutional Review Board of CHA University. To obtain the urine cells, approximately 100e200 ml of midstream urine was collected from an adult and centrifuged at 400 g for 30 min to pellet the cells in the collected urine sample. The cell pellet was washed twice with 30 ml of 1x PBS containing 1 antibiotic-antimycotic solution (Invitrogen, Carlsbad, CA, USA) and subsequently resuspended in 5 ml of culture medium containing equal volumes of a modified Renal Epithelial Cell Growth Medium (REGM; Lonza, Walkersville, MD, USA) and MesenGro® medium (StemRD, Burlingame, CA, USA) (1:1, v/v). The modified REGM used in this study contained human serum (SigmaeAldrich, St Louis, MO, USA) instead of fetal bovine serum (FBS; Invitrogen); therefore, it was free of animal components. The resuspended cells were seeded in 6-cm dishes coated with human gelatin (Fibrogen, San Francisco, CA, USA) and cultured; the medium was changed every 2 or 3 days. To obtain the ADSCs, a small piece of adipose tissue (approximately 0.125 cm3) was minced with a blade, transferred into a 15-ml Falcon tube (BD Biosciences, San Jose, CA, USA) containing an equal volume of xeno-free collagenase (0.075%) (Worthington Biochemical, Lakewood, NJ, USA), and incubated for 30 min in a 37 C water bath with occasional mixing. The cell pellet was collected by centrifugation at 1200 rpm for 5 min, resuspended in 10 ml of xeno-free MesenGro® medium (StemRD), and filtered through a cell strainer with a 100-mm nylon mesh (BD Biosciences). The filtrate was collected in a 50-mlFalcon tube (BD Biosciences), washed once with 10 ml of 1 PBS (Invitrogen), and finally resuspended in 10 ml of MesenGro® medium prior to seeding into a 10-cm dish for culture. 2.2. Proliferation and viability of the urine cells and ADSCs The growth rates of the urine cells and ADSCs were measured by counting the number of cells on days 1, 3, 5, and 7 after seeding; initially, 5 104 cells were seeded in each well of a 6-well plate. Cell viability was also measured on the same day via a water-soluble tetrazolium salt (WST) method using the EZ-Cytox Cell Viability Assay Kit (Daeil Lab Service, Seoul, Korea) according to the manufacturer's protocol. The non-xeno-free medium used for culturing the urine cells contained a 1:1 ratio of DMEM/Ham's F12 media (Hyclone, Logan, UT, USA), 10% FBS, SingleQuot Kit CC-4217 REGM (Lonza, Walkersville, MD, USA), and 1 amphotericin B. 2.3. The generation of footprint- and xeno-free iPSCs from urine cells and ADSCs A total of 1 106 urine cells and ADSCs were resuspended in a single-cell suspension using trypsin and were electroporated with 3 reprogramming plasmids (pCXLE-hOCT4-shp53, pCXLE-hSK, pCXLE-hUL) (Addgene, Cambridge, MA, USA) using the Neon Transfection System (Invitrogen). The transfected cells were seed on a 6-cm dish and were cultured in their respective xeno-free culture media for 5 days. The cells were then replated at a density of 1e3 104 cells per well in a 6-well plate pre-coated with vitronectin (BD Biosciences) and cultured in their xeno-free primary cell media for 2 more days; finally, the medium was changed to an ECM-based xeno-free hPSC medium until all the iPSC colonies were harvested. The composition of the ECM-based xeno-free hPSC medium was previously described [22]. To coat a 6-well plate, we added 1 ml of vitronectin solution (5 mg/ml in 1X PBS) to each well and incubated the plate at room temperature for at least 1 h. The vitronectin solution was removed by aspirating before plating the cells. 2.4. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) Total RNA was prepared using a NucleoSpin RNA II kit (MACHEREY-NAGEL GmbH, Duren, Germany) according to the manufacturer's protocol. One microgram of RNA was converted into complementary DNA using the ReverTra Ace qPCR-RT Kit (Toyobo, Osaka, Japan). qRT-PCR was performed using the StepOnePlus™ Real-Time PCR System (Invitrogen). The following qRT-PCR conditions were used: (1) denaturation at 95 C for 40 s, (2) primer annealing at 55e63 C for 30 s, and (3) extension at 72 C for 1 min. These steps were cycled 40 times, followed by a final polymerization step at 72 C for 10 min. The primers used in qRT-PCR are listed in Supplementary Table 2. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a normalization control. Semi-quantitative RT-PCR was performed using a T100 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) using the same conditions described above, except
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that the number of cycles ranged from 20 to 35 depending on the abundance of the target mRNA. 2.5. Pluripotency tests of iPSCs To test the pluripotency in vitro, the iPSC colonies were fragmented and grown in suspension for 15 days in embryoid body (EB) medium (DMEM/F12, 10% Knockout™SR, 1% NEAA, 0.1 mM b-mercaptoethanol, and 1% penicillin/streptomycin) (all from Invitrogen). The EBs were then cultured on matrigel-coated dishes for 15 days in spontaneous differentiation medium (DMEM/F12, 10% FBS, 1% NEAA, 0.1 mM bmercaptoethanol, and 1% penicillin/streptomycin) (all from Invitrogen) and subjected to immunostaining for markers of all three germ layers. Pluripotency was examined in vivo by determining the ability of the cells to form teratomas in NOD/SCID mice. To this end, colonies containing approximately 2 106 cells were collected and injected into the muscles of NOD/SCID mice. After approximately 9 weeks, the teratomas were examined for the presence of all three germ layer structures using hematoxylin and eosin staining. 2.6. Karyotyping analysis A G-band karyotyping of the iPSCs was performed in the Samkwang Medical Laboratories (Smlab, Seoul, Korea). 2.7. DNA Fingerprinting To confirm the identity of the iPSC clones derived from both the urine cells and ADSCs, we performed DNA fingerprinting analyses at the DNA Sequencing Core Facility of Korea Gene Information Center (KGIC, Seoul, Korea). 2.8. Bisulfite sequencing Genomic DNA was prepared from the urine cells, ADSCs, hESCs, and several iPSC lines using the DNeasy Tissue Kit (Qiagen, Hilden, Germany), and the DNA was then treated with an EpiTect Bisulfite Kit (Qiagen) according to the manufacturer's instructions. The methylation patterns of the 50 upstream regions of the Oct4 and nanog genes were subsequently analyzed using DNA sequencing (Solgent, Daejeon, Korea). The primers used in the bisulfite sequencing reactions are listed in Supplementary Table 2. 2.9. Gene expression profiling Total RNA was isolated using the NucleoSpin RNA II kit (MACHEREY-NAGEL GmbH) according to the manufacturer's suggestions, and 2 mg of total RNA was utilized for a genome-wide gene expression profiling experiment using the Illumina array (Illumina, San Diego, CA, USA) at Macrogen (Macrogen, Seoul, Korea).
3. Results 3.1. Culture of human somatic cells in xeno-free culture conditions To produce xeno-free iPSCs, the primary cells used for iPSC generation should be cultured in xeno-free culture conditions from the time of biopsy. In our study, we prepared and cultured human urine-derived cells in modified REGM:MesenGro® (1:1, v/v) medium. ADSCs, another cells used in this study, were cultured in a commercially available xeno-free medium, MesenGro® medium. In their respective xeno-free media, the number of urine cells tended to be lower than that of the ADSCs (Fig. 1A) at multiple time points (days 1, 3, 5, and 7) after seeding, indicating that the urine cells grew more slowly than the ADSCs. This result was supported by the data obtained from the WST assay (Fig. 1B). The urinederived cells displayed both elongated and epithelial cell-like morphologies, while the ADSCs displayed a spindle shape, which is a typical morphology of mesenchymal stem cells (Supplementary Fig. 1). The different proliferation rates of the urine cells and ADSCs in their xeno-free culture media were still evident after the electroporation of plasmids encoding the reprogramming genes (Fig. 1C and D). Therefore, the urine cells grew slower than ADSCs both before (Fig. 1A and B) and after (Fig. 1C and D) electroporation. Next, we wanted to confirm that the xeno-free culture media formulated in this study efficiently promoted the growth of the urine cells and ADSCs. To this end, we compared the growth of the cells in their corresponding xeno-free media with the non-xenofree media previously used by other groups, including DMEM/
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Fig. 1. Proliferation of human urine cells and ADSCs under xeno-free conditions. (A) The cells at p3 in each well of a 6-well plate were counted on days 1, 3, 5, and 7 after seeding; all wells were initially seeded with 5 104 cells/well. (B) The WST assay was performed to confirm the results shown in (A). (C) The urine cells and ADSCs were electroporated with three plasmids encoding reprogramming factors, and the cells were counted at several time points after electroporation. (D) The WST assay was performed to confirm the results shown in (C). (E) The proliferation rates of urine cells and ADSCs were examined under a variety of culture (xeno-free and non-xeno-free) conditions. (**P < 0.01).
Ham's F12 (1:1, v/v) containing 10% FBS and SingleQuot Kit CC-4217 REGM (for the urine cells) [21] and a-MEM containing 10% FBS (for the ADSCs) [23]. Our results demonstrated that the xeno-free media used in this study could indeed efficiently promote the growth of both the urine cells and ADSCs (Fig. 1E). In summary, both the urine cells and the ADSCs were cultured efficiently in their respective xeno-free media, although the urine cells showed a lower proliferative rate than the ADSCs. Thus, xenofree media would be suitable for the generation of xeno-free iPSCs.
total cell population between urine cells (~27%) and ADSCs (~13%) became obvious at passage 7 (Fig. 2A and B, middle panels). Strikingly, at passage 15, most of the urine cells (~98%) were annexin Vpositive, while only 17% of the ADSCs were annexin V-positive (Fig. 2A and B, bottom panels). These results indicated that the urine cells apparently senesce much earlier than the ADSCs in the xeno-free culture media used in this study. This result suggested that the use of low passage urine cells would be recommended for iPSC generation.
3.2. Cell death of urine cells and ADSCs at different passage numbers
3.3. Generation of xeno- and integration-free iPSCs from urine cells and ADSCs
We further examined whether the urine-derived cells and ADSCs could be maintained for an extended time period in their xeno-free media. The percentage of apoptotic (annexin V-positive) cells was comparably low (approximately 7e8% of the total cells) at low passages (~P3) for both cell types (Fig. 2A and B, top panels). However, the difference in the percentage of apoptotic cells in the
For therapeutic applications, the generation of strictly xeno- and integration-free iPSCs is essential. Therefore, we examined whether such xeno- and integration-free iPSCs could be generated by elaborately combining the EBNA1/OriP-plasmid transfection method [3] with the ECM(vitronectin)-based xeno-free hPSC culture conditions established in our laboratory [22].
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Fig. 2. The percentage of annexin V-positive cells was measured for the urine cells and ADSCs at passages 3, 7, and 15. The cells were stained with Hoechst 33342, Annexin V-FITC, and propidium iodide (PI). Hoechst 33342 stained the total cell population (left panels), while Annexin V stained the apoptotic and necrotic cells. Early apoptotic cells exclude PI (Annexin V-positive and PI-negative cells), whereas late-stage apoptotic and necrotic cells are positively stained for both Annexin V and PI (middle panels). Histograms (right panels) show the Annexin V-FITC intensity of the cell population. (A) Urine cells. (B) ADSCs.
First, the primary cells were electroporated with the three EBNA1/OriP-based plasmids encoding the 7 reprogramming factors (Oct4, Sox2, Lin28, L-Myc, Klf4, p53 shRNA, and SV40LT), and the resulting cells were then maintained under the ECM-based xenofree hPSC culture conditions until ESC-like colonies were formed. The ESC-like colonies were counted on days 18 and 23 after electroporation, and colonies were selected for expansion under the ECM-based xeno-free hPSC culture condition (Fig. 3A). Evidently, more colonies were formed from the ADSCs than the urine cells on days 18 and 23 after electroporation (~0.3% from the urine cells and ~0.4% from the ADSCs on day 23) (Fig. 3B). In summary, the xeno-free vitronectin-based hPSC culture condition was compatible with the episomal plasmid transfection method for the generation of xeno- and footprint-free iPSCs from both urine cells and ADSCs. Furthermore, the efficiency of iPSC generation via this method appeared to be higher than what was previously reported using the retroviral gene delivery/feeder-based culture system. 3.4. Gene expression profiles of the iPSCs derived from urine cells and ADSCs For detailed characterization, we chose two lines each from the iPSC lines that originated from the urine cells and the ADSCs (UiPSC1 and 2 and AiPSC1 and 2, respectively). First, we used DNA fingerprinting analysis to confirm that the iPSC clones we chose
were indeed derived from their original primary cells (either urine cells or ADSCs) (Supplementary Table 1). The iPSC lines derived from both cell types expressed multiple representative pluripotent cell markers, including Oct4, Sox2, SSEA4, Tra1-60, Tra1-81, Rex1, DNMT3B, and ZIC3, as detected by either immunostaining (Fig. 4A and B) or quantitative RT-PCR (Fig. 4C). Additionally, all the iPSC lines expressed a low level of differentiation markers for the three germ lineages, such as SOX1 and PAX6 (ectoderm), GATA2 and Brachyury (mesoderm), and AFP and SOX17 (endoderm) (Fig. 4D). Furthermore, genome-wide gene expression profiling analysis revealed a similar gene expression pattern between the iPSCs derived from urine cells and those derived from ADSCs (Fig. 5E). Scatter plot, heatmap, and hierarchical clustering of the genome-wide gene expression profiles all indicated a close correlation between UiPSC1, AiPSC1, and H9-hESC lines (Fig. 5AeG). PluriTest, a gene expression-based bioinformatic diagnostic test for evaluating pluripotency [24], showed that the iPSCs derived from urine cells and ADSCs were indeed fully reprogrammed pluripotent stem cells (Fig. 5H). 3.5. Characterization of the iPSCs derived from urine cells and ADSCs Bisulfide sequencing analysis revealed largely demethylated patterns in the Oct4 and Nanog promoters of the iPSC lines derived from both urine cells and ADSCs (Fig. 6A). On the contrary, the Oct4
Fig. 3. Generation of iPSCs from urine cells and ADSCs. (A) Schedule of the experiment. (B) ESC-like colonies were counted at day 18 and 23 post-electroporation. The colonies were immunostaining with an antibody targeting the undifferentiated cell marker Tra1-60. (**P < 0.01).
Fig. 4. Characterization of iPSCs generated from urine cells and ADSCs. The iPSC colonies that were generated and cultured for 23e25 passages were subjected to immunostaining (A, B) and real-time RT-PCR (C, D) to examine the undifferentiated cell markers Oct4, Sox2, SSEA4, Tra1-60, and Tra1-81. (A) Urine cell-derived iPSCs. (B) ADSC-derived iPSCs. A DIC image of a single colony is also shown. All the colonies expressed alkaline phosphatase, a marker for pluripotent stem cells. (C) Quantitative real-time RT-PCR was performed to detect the expression of the undifferentiated cell markers Oct4, nanog, Sox2, Rex1, DNMT3B, and Zic3. (D) Quantitative real-time RT-PCR was performed to detect the expression of the representative markers for the ectoderm (SOX1, PAX6), mesoderm (GATA6, Brachyury), and endoderm (AFP, SOX17) lineages. (**P < 0.01) Scale bar ¼ 100 mm.
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Fig. 5. Genome-wide gene expression profile analysis. (A-E) Scatter plot of gene expression profiles for iPSCs, hESCs, and primary cells. (F) Hierarchical clustering of the iPSCs, hESCs, and primary cells based on their gene expression patterns. (G) A heatmap of the microarray data from the iPSCs, hESCs, and primary cells. The genes shown in green are those that were upregulated, whereas the genes shown in red are those that were downregulated. (H) PluriTest analysis of the iPSCs, hESCs, and primary cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
and Nanog promoters of the primary cellsdboth urine cells and ADSCsdwere heavily methylated (Fig. 6A). These results were consistent with the iPSC-specific expression of the Oct4 and Nanog genes. The iPSCs retained pluripotency because these cells were able to differentiate into cells from all three germ layers (Fig. 6B and C). Gbanding chromosomal analysis indicated that there were no gross chromosomal aberrations in the iPSC lines generated and cultured in the ECM-based xeno-free hPSC medium (Supplementary Fig. 2A). Importantly, PCR analysis using episomal plasmid-specific primer pairs clearly demonstrated that the episomal plasmids were not integrated into the host chromosomes (Supplementary Fig. 2B). Taken together, our results reported that xeno- and footprintfree iPSCs were efficiently generated from urine cells, which can
be obtained in a non-invasive manner. Additionally, we showed that our combined method worked with other primary cells, such as ADSCs, which can also be easily acquired from patients. Therefore, our ECM-based (feeder-independent) method of generating xeno- and footprint-free iPSCs established in this study would be of great clinical value by providing an unlimited number of autologous or immune-compatible cell sources for future iPSC-based cell replacement therapy. 4. Discussion Clinically applicable, safe iPSCs should ideally be generated using chromosome integration-free methods under xeno-free culture conditions. Until now, several methods for producing integrationfree iPSCs have been developed; among these, the EBNA1/OriPbased plasmid method [1e3], the mRNA transfection method [4],
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Fig. 6. DNA methylation of Oct4 and nanog promoters and pluripotency analysis in vitro and in vivo. (A) Bisulfite sequencing analysis showed that the Oct4 and nanog promoters in the iPSCs were largely demethylated, similar to what was observed in hESCs. (B) In vitro pluripotency test. The iPSCs spontaneously differentiated, and the expression levels of representative markers of the ectoderm (nestin, beta-III tubulin), mesoderm (smooth muscle actin, PECAM), and endoderm (a-fetoprotein, FoxA2) lineages were examined. (C) The derivatives of the three germ layers were detected in teratomas, which formed approximately 9 weeks after iPSC administration (2 106 cells) into NOD/SCID mice. Scale bar ¼ 100 mm.
and the Sendai viral method [5] are commonly used. Therefore, an ECM-based xeno-free hPSC culture condition that supports the efficient generation of iPSCs in combination with the aforementioned integration-free methods is greatly in demand. For the purpose of iPSC-based cell therapy, we sought to generate xeno- and integration-free iPSCs using the ECM-based (feeder-independent) xeno-free hPSC culture medium we established. As an ECM, we used human vitronectin that was purified from human plasma, and the xeno-free medium was formulated around a protein kinase C inhibitor, which is critical for the maintenance of stemness [22]. Although this xeno-free hPSC culture
system was shown to support the efficient generation of iPSCs using the retrovirus-mediated method, its compatibility with an integration-free method for the generation of iPSCs had not been tested. This is a very important issue to explore for future cell replacement therapeutic approaches. Therefore, in this study, we tested whether integration- and xeno-free iPSCs were generated efficiently by elaborately combining the EBNA1/OriP-based plasmid method and the ECM-based xeno-free hPSC culture medium. The choice of cell sources for iPSC generation is also an important factor to consider. Ideal cell sources are cells that can be obtained from patients with no or minimal invasiveness and can also
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be reprogrammed into iPSCs with high efficiency. In this study, we tested whether xeno- and integration-free iPSCs can be easily generated from urine cells. Urine cells are a heterogeneous population of cells comprising renal tube epithelial cells, urothelial cells, fibroblast-like cells, and urine-derived adult stem cells (i.e., mesenchymal stem cells) [25]. To generate strictly xeno-free iPSCs, we grew the primary cells, urine cells and ADSCs, under xeno-free conditions starting from biopsy. Although a xeno-free primary cell culture medium for ADSCs was commercially available, no xeno-free medium was available for the urine cells. Therefore, we formulated a new xenofree culture medium for these cells by replacing bovine serum albumin with human serum albumin from REGM, and we mixed the modified REGM with MesenGro® medium at a 1:1 ratio (v/v). The urine cells and the ADSCs grew well in their respective xeno-free media; however, the ADSCs grew faster than the urine cells. Intriguingly, the proportion of dead cells among the total cells was low (7e8%) at early passages (P3) in both cell types, although the urine cells tended to die much faster than the ADSCs as the passage number increased (27% vs. 13% at P7 and 98% vs. 17% at P15) (Fig. 2). It is currently not clear whether this result was caused by an intrinsic characteristic of the urine-derived cells and ADSCs or by the xeno-free culture conditions used for their culture. We believe that the former possibility is more likely because the urine cells tended to enter senescence much earlier than the ADSCs, regardless of which media were used for culture (data not shown); however, this would not affect the generation of iPSCs because we mostly use cells with low passage numbers (less than 4) for iPSC generation. The results of this study showed that the number of iPSCs generated from the adipose-derived stromal cells was higher than the number derived from the urine cells (0.38% vs. 0.29% at day 23). The efficiency of ADSC-derived iPSC generation using our integration- and xeno-free method appeared to be higher than that reported in a previous paper describing the generation of AiPSCs using retroviral mediated gene delivery (0.2% on MEFs and 0.01e0.03% on Matrigel) [18]. The efficiency of UiPSC generation was within the range previously reported for UiPSC generation using the retroviral method on MEF feeder cells (0.01e4%) [21]. It was generally thought that feeder-free culture conditions were less efficient for iPSC generation than the feeder-dependent culture system [18]. In this regard, the apparently high efficiency of iPSC generation in the feeder-free conditions in our study may reflect the intrinsic enhancing activity of our ECM-based xeno-free hPSC culture system, which was previously described to be significantly higher than the conventional MEF-dependent method [22]. The efficiency of iPSC generation may also be affected by the proliferation rate of the primary cells that are being used. Although there are reports arguing that a negative correlation exists between the growth rate and the iPSC formation efficiency [26], this issue remains unclear due to multiple contradictory reports [27,28]. At present, it is not clear whether the more efficient iPSC formation of ADSCs compared to urine cells is caused by the higher proliferative activity or any intrinsic property of ADSCs. In this study, we have demonstrated two important results regarding the generation of xeno- and integration-free iPSCs: (1) Our ECM-based xeno-free hPSC culture system supported the efficient generation of integration-free iPSCs from both urine cells and ADSCs, indicating that the xeno-free hPSC culture system functions well with the integration-free iPSC generation method. (2) Early passage urine cells are good cell sources for iPSC generation due to the non-invasive nature of collection and their high reprogramming efficacy. Taken together, this study reported the efficient generation of xeno-free and footprint-free iPSCs from urine cells, which can be obtained non-invasively, and also provides an efficient and
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convenient platform for the generation of therapeutically applicable iPSCs for cell therapy. 5. Conclusions In this study, we reports the efficient ECM-based (feeder-independent) generation of integration- and xeno-free iPSCs from easily obtainable urine cells and hence, provides a platform that can be used to generate iPSCs for cell replacement therapy in the foreseeable future. Author contributions D.-Y. H. designed the project and wrote the manuscript. K.-I. L and H.-T. K. equally contributed by performed most experiments. Acknowledgments This work was supported by grants from the Stem Cell Research Program (2010-0020350) and 2012M3A9C7050130 from the MSIP, and A120254-1201-0000200 from the Ministry of Health & Welfare, Korea. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.05.059. References [1] Jia F, Wilson KD, Sun N, Gupta DM, Huang M, Li Z, et al. A nonviral minicircle vector for deriving human iPS cells. Nat Meth 2010;7:197e9. [2] Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin II , et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science (New York, Ny) 2009;324:797e801. [3] 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 Meth 2011;8:409e12. [4] Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F, et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 2010;7:618e30. [5] Fusaki N, Ban H, Nishiyama A, Saeki K, Hasegawa M. Efficient induction of transgene-free human pluripotent stem cells using a vector based on sendai virus, an RNA virus that does not integrate into the host genome. proceedings of the Japan academy series B. Phys Biol Sci 2009;85:348e62. [6] Martin MJ, Muotri A, Gage F, Varki A. Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat Med 2005;11:228e32. [7] Heiskanen A, Satomaa T, Tiitinen S, Laitinen A, Mannelin S, Impola U, et al. Nglycolylneuraminic acid xenoantigen contamination of human embryonic and mesenchymal stem cells is substantially reversible. Stem Cells (Dayt Ohio) 2007;25:197e202. [8] Hu W, Yan Q, Fang Y, Qiu Z, Zhang S. Generation of human induced pluripotent stem cells with non-integrating episomal vectors and xeno-free culture system. J Anim Veterinary Adv 2013;12:1114e22. [9] Nakagawa M, Taniguchi Y, Senda S, Takizawa N, Ichisaka T, Asano K, et al. A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells. Sci Rep 2014;4:3594. [10] Fukusumi H, Shofuda T, Kanematsu D, Yamamoto A, Suemizu H, Nakamura M, et al. Feeder-free generation and long-term culture of human induced pluripotent stem cells using pericellular matrix of decidua derived mesenchymal cells. PloS One 2013;8:e55226. [11] 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;131:861e72. [12] Takenaka C, Nishishita N, Takada N, Jakt LM, Kawamata S. Effective generation of iPS cells from CD34þ cord blood cells by inhibition of p53. Exp Hematol 2010;38:154e62. [13] Haase A, Olmer R, Schwanke K, Wunderlich S, Merkert S, Hess C, et al. Generation of induced pluripotent stem cells from human cord blood. Cell Stem Cell 2009;5:434e41. [14] Giorgetti A, Montserrat N, Aasen T, Gonzalez F, Rodriguez-Piza I, Vassena R, et al. Generation of induced pluripotent stem cells from human cord blood using OCT4 and SOX2. Cell Stem Cell 2009;5:353e7. [15] Loh YH, Hartung O, Li H, Guo C, Sahalie JM, Manos PD, et al. Reprogramming of T cells from human peripheral blood. Cell Stem Cell 2010;7:15e9.
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[16] Staerk J, Dawlaty MM, Gao Q, Maetzel D, Hanna J, Sommer CA, et al. Reprogramming of human peripheral blood cells to induced pluripotent stem cells. Cell Stem Cell 2010;7:20e4. [17] Seki T, Yuasa S, Oda M, Egashira T, Yae K, Kusumoto D, et al. Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells. Cell Stem Cell 2010;7:11e4. [18] Sun N, Panetta NJ, Gupta DM, Wilson KD, Lee A, Jia F, et al. Feeder-free derivation of induced pluripotent stem cells from adult human adipose stem cells. Proc Natl Acad Sci U S A 2009;106:15720e5. [19] Kim JB, Greber B, Arauzo-Bravo MJ, Meyer J, Park KI, Zaehres H, et al. Direct reprogramming of human neural stem cells by OCT4. Nature 2009;461: 649e53. [20] Aasen T, Raya A, Barrero MJ, Garreta E, Consiglio A, Gonzalez F, et al. Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat Biotechnol 2008;26:1276e84. [21] Zhou T, Benda C, Duzinger S, Huang Y, Li X, Li Y, et al. Generation of induced pluripotent stem cells from urine. J Am Soc Nephrol: JASN 2011;22:1221e8.
[22] Kim HT, Lee KI, Kim DW, Hwang DY. An ECM-based culture system for the generation and maintenance of xeno-free human iPS cells. Biomaterials 2013;34:1041e50. [23] Jeon ES, Moon HJ, Lee MJ, Song HY, Kim YM, Cho M, et al. Cancer-derived lysophosphatidic acid stimulates differentiation of human mesenchymal stem cells to myofibroblast-like cells. Stem Cells (Dayt Ohio) 2008;26:789e97. [24] Muller FJ, Schuldt BM, Williams R, Mason D, Altun G, Papapetrou EP, et al. A bioinformatic assay for pluripotency in human cells. Nat Meth 2011;8:315e7. [25] Benda C, Zhou T, Wang X, Tian W, Grillari J, Tse HF, et al. Urine as a source of stem cells. Adv Biochem Eng Biotechnol 2013;129:19e32. [26] Xu Y, Wei X, Wang M, Zhang R, Fu Y, Xing M, et al. Proliferation rate of somatic cells affects reprogramming efficiency. J Biol Chem 2013;288:9767e78. [27] Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, et al. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol 2008;26:101e6. [28] Wernig M, Meissner A, Cassady JP, Jaenisch R. c-Myc is dispensable for direct reprogramming of mouse fibroblasts. Cell Stem Cell 2008;2:10e2.
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Footprint- and xeno-free human iPSCs derived from urine cells using extracellular matrix-based culture conditions Kang-In Lee 1, Hyeong-Taek Kim 1, Dong-Youn Hwang* Department of Biomedical Sciences, CHA University, Seongnam, Kyeonggido 463-840, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 March 2014 Accepted 21 May 2014 Available online 30 June 2014
The efficient generation of integration- and xeno-free iPSCs is a prerequisite for their use in clinical applications. Furthermore, non-invasiveness of somatic cell acquisition for iPSC generation is another factor to consider. In this study, we established a practical, simple, and convenient method to generate integration- and xeno-free iPSCs from urine cells which can be obtained in a non-invasive manner. Our method was based on extracellular matrix-based xeno-free iPSC culture condition and episomal transfection, and worked efficiently with both urine cells and adipose-derived stromal cells (ADSCs). To obtain strictly xeno-free iPSCs, we also formulated a new xeno-free culture medium for primary urine cells. Intriguingly, urine cells displayed slower growth, and more dramatic increase in apoptosis at high passage numbers than ADSCs. However, urine cells at low passage (
Keywords: Integration-free Xeno-free Urine cells Induced pluripotent stem cells Cell therapy
1. Introduction One of the most valuable advantages of human induced pluripotent stem cells (iPSCs) is their potential for use in immunecompatible cell replacement therapy. For cell therapy purposes, the generation of footprint- and xeno-free iPSCs is one of the major concerns that remains to be addressed. In this regard, rapid progress has been made in reprogramming somatic cells without the insertion of transgenes into the chromosomes. Until now, the overexpression of reprogramming factors using EBNA1/OriP (Epstein-Barr nuclear antigen Origin of plasmid replication)-based plasmids [1e3], mRNAs [4], and Sendai viruses [5] has attracted significant attention as methods that generate footprint-free iPSCs. Another important safety-related issue is the establishment of xeno-free conditions for both the derivation and expansion of the iPSCs. Conventionally, human pluripotent stem cells (hPSCs) were cultured on mouse embryonic fibroblast feeder cells using culture media containing animal-derived components. The mouse feeder cell-dependent culture system faced potential risks, including the transmission of non-human pathogens to humans and immune
* Corresponding author. Tel.: þ82 31 8017 9411; fax: þ82 31 750 9447. E-mail address: [email protected] (D.-Y. Hwang). 1 Both authors contributed equally to this work. http://dx.doi.org/10.1016/j.biomaterials.2014.05.059 0142-9612/© 2014 Elsevier Ltd. All rights reserved.
rejection problems elicited by contamination from non-human antigens [6,7]. Therefore, an extracellular matrix (ECM)-based feeder-independent culture system is of great value to produce and culture xeno-free human iPSCs. With the initiation of a clinical trial using iPSCs for the treatment of macular degeneration, the development of method to generate xeno- and footprint-free iPSCs became more important than ever. In this regard, recent reports demonstrated successful generation of iPSCs using xeno-free media in combination with xeno-free ECMs, such as vitronectin [8], laminin [9], and pericellular matrix of decidua-derived mesenchymal stem cells [10]. Another important issue to consider for the generation of iPSCs from patients is the source of somatic cells that are used for iPSC generation. A variety of cell types, including dermal fibroblasts [11], bone marrow CD34 þcells [12], cord blood cells [13,14], peripheral blood cells [15e17], adipose-derived stromal cells [18], neural stem cells [19], and keratinocytes [20], have been used to generate iPSCs. There is a growing consensus that an ideal cell source for iPSC generation should be acquired easily and noninvasively, and the efficiency of iPSC generation should be considerably high. Recently, urine cells were proposed as a promising cell source for iPSC generation, as these cells can be obtained in a non-invasive manner [21]. In this study, we sought to establish an efficient method to generate integration- and xeno-free iPSCs by elaborately combining
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an ECM-based feeder-independent/xeno-free hPSC culture system [22] and episomal vector transfection method. Furthermore, we examined if the combined iPSC-generating system functioned for urine cells which are non-invasively obtainable cells in the human body. The generation of integration- and xeno-free iPSCs from urine cells would provide a convenient and easy-to-access platform for iPSC-based cell replacement therapies in the near future. 2. Materials and methods 2.1. The isolation and culture of urine cells and adipose-derived stromal cells (ADSCs) This study was approved by the Institutional Review Board of CHA University. To obtain the urine cells, approximately 100e200 ml of midstream urine was collected from an adult and centrifuged at 400 g for 30 min to pellet the cells in the collected urine sample. The cell pellet was washed twice with 30 ml of 1x PBS containing 1 antibiotic-antimycotic solution (Invitrogen, Carlsbad, CA, USA) and subsequently resuspended in 5 ml of culture medium containing equal volumes of a modified Renal Epithelial Cell Growth Medium (REGM; Lonza, Walkersville, MD, USA) and MesenGro® medium (StemRD, Burlingame, CA, USA) (1:1, v/v). The modified REGM used in this study contained human serum (SigmaeAldrich, St Louis, MO, USA) instead of fetal bovine serum (FBS; Invitrogen); therefore, it was free of animal components. The resuspended cells were seeded in 6-cm dishes coated with human gelatin (Fibrogen, San Francisco, CA, USA) and cultured; the medium was changed every 2 or 3 days. To obtain the ADSCs, a small piece of adipose tissue (approximately 0.125 cm3) was minced with a blade, transferred into a 15-ml Falcon tube (BD Biosciences, San Jose, CA, USA) containing an equal volume of xeno-free collagenase (0.075%) (Worthington Biochemical, Lakewood, NJ, USA), and incubated for 30 min in a 37 C water bath with occasional mixing. The cell pellet was collected by centrifugation at 1200 rpm for 5 min, resuspended in 10 ml of xeno-free MesenGro® medium (StemRD), and filtered through a cell strainer with a 100-mm nylon mesh (BD Biosciences). The filtrate was collected in a 50-mlFalcon tube (BD Biosciences), washed once with 10 ml of 1 PBS (Invitrogen), and finally resuspended in 10 ml of MesenGro® medium prior to seeding into a 10-cm dish for culture. 2.2. Proliferation and viability of the urine cells and ADSCs The growth rates of the urine cells and ADSCs were measured by counting the number of cells on days 1, 3, 5, and 7 after seeding; initially, 5 104 cells were seeded in each well of a 6-well plate. Cell viability was also measured on the same day via a water-soluble tetrazolium salt (WST) method using the EZ-Cytox Cell Viability Assay Kit (Daeil Lab Service, Seoul, Korea) according to the manufacturer's protocol. The non-xeno-free medium used for culturing the urine cells contained a 1:1 ratio of DMEM/Ham's F12 media (Hyclone, Logan, UT, USA), 10% FBS, SingleQuot Kit CC-4217 REGM (Lonza, Walkersville, MD, USA), and 1 amphotericin B. 2.3. The generation of footprint- and xeno-free iPSCs from urine cells and ADSCs A total of 1 106 urine cells and ADSCs were resuspended in a single-cell suspension using trypsin and were electroporated with 3 reprogramming plasmids (pCXLE-hOCT4-shp53, pCXLE-hSK, pCXLE-hUL) (Addgene, Cambridge, MA, USA) using the Neon Transfection System (Invitrogen). The transfected cells were seed on a 6-cm dish and were cultured in their respective xeno-free culture media for 5 days. The cells were then replated at a density of 1e3 104 cells per well in a 6-well plate pre-coated with vitronectin (BD Biosciences) and cultured in their xeno-free primary cell media for 2 more days; finally, the medium was changed to an ECM-based xeno-free hPSC medium until all the iPSC colonies were harvested. The composition of the ECM-based xeno-free hPSC medium was previously described [22]. To coat a 6-well plate, we added 1 ml of vitronectin solution (5 mg/ml in 1X PBS) to each well and incubated the plate at room temperature for at least 1 h. The vitronectin solution was removed by aspirating before plating the cells. 2.4. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) Total RNA was prepared using a NucleoSpin RNA II kit (MACHEREY-NAGEL GmbH, Duren, Germany) according to the manufacturer's protocol. One microgram of RNA was converted into complementary DNA using the ReverTra Ace qPCR-RT Kit (Toyobo, Osaka, Japan). qRT-PCR was performed using the StepOnePlus™ Real-Time PCR System (Invitrogen). The following qRT-PCR conditions were used: (1) denaturation at 95 C for 40 s, (2) primer annealing at 55e63 C for 30 s, and (3) extension at 72 C for 1 min. These steps were cycled 40 times, followed by a final polymerization step at 72 C for 10 min. The primers used in qRT-PCR are listed in Supplementary Table 2. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a normalization control. Semi-quantitative RT-PCR was performed using a T100 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) using the same conditions described above, except
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that the number of cycles ranged from 20 to 35 depending on the abundance of the target mRNA. 2.5. Pluripotency tests of iPSCs To test the pluripotency in vitro, the iPSC colonies were fragmented and grown in suspension for 15 days in embryoid body (EB) medium (DMEM/F12, 10% Knockout™SR, 1% NEAA, 0.1 mM b-mercaptoethanol, and 1% penicillin/streptomycin) (all from Invitrogen). The EBs were then cultured on matrigel-coated dishes for 15 days in spontaneous differentiation medium (DMEM/F12, 10% FBS, 1% NEAA, 0.1 mM bmercaptoethanol, and 1% penicillin/streptomycin) (all from Invitrogen) and subjected to immunostaining for markers of all three germ layers. Pluripotency was examined in vivo by determining the ability of the cells to form teratomas in NOD/SCID mice. To this end, colonies containing approximately 2 106 cells were collected and injected into the muscles of NOD/SCID mice. After approximately 9 weeks, the teratomas were examined for the presence of all three germ layer structures using hematoxylin and eosin staining. 2.6. Karyotyping analysis A G-band karyotyping of the iPSCs was performed in the Samkwang Medical Laboratories (Smlab, Seoul, Korea). 2.7. DNA Fingerprinting To confirm the identity of the iPSC clones derived from both the urine cells and ADSCs, we performed DNA fingerprinting analyses at the DNA Sequencing Core Facility of Korea Gene Information Center (KGIC, Seoul, Korea). 2.8. Bisulfite sequencing Genomic DNA was prepared from the urine cells, ADSCs, hESCs, and several iPSC lines using the DNeasy Tissue Kit (Qiagen, Hilden, Germany), and the DNA was then treated with an EpiTect Bisulfite Kit (Qiagen) according to the manufacturer's instructions. The methylation patterns of the 50 upstream regions of the Oct4 and nanog genes were subsequently analyzed using DNA sequencing (Solgent, Daejeon, Korea). The primers used in the bisulfite sequencing reactions are listed in Supplementary Table 2. 2.9. Gene expression profiling Total RNA was isolated using the NucleoSpin RNA II kit (MACHEREY-NAGEL GmbH) according to the manufacturer's suggestions, and 2 mg of total RNA was utilized for a genome-wide gene expression profiling experiment using the Illumina array (Illumina, San Diego, CA, USA) at Macrogen (Macrogen, Seoul, Korea).
3. Results 3.1. Culture of human somatic cells in xeno-free culture conditions To produce xeno-free iPSCs, the primary cells used for iPSC generation should be cultured in xeno-free culture conditions from the time of biopsy. In our study, we prepared and cultured human urine-derived cells in modified REGM:MesenGro® (1:1, v/v) medium. ADSCs, another cells used in this study, were cultured in a commercially available xeno-free medium, MesenGro® medium. In their respective xeno-free media, the number of urine cells tended to be lower than that of the ADSCs (Fig. 1A) at multiple time points (days 1, 3, 5, and 7) after seeding, indicating that the urine cells grew more slowly than the ADSCs. This result was supported by the data obtained from the WST assay (Fig. 1B). The urinederived cells displayed both elongated and epithelial cell-like morphologies, while the ADSCs displayed a spindle shape, which is a typical morphology of mesenchymal stem cells (Supplementary Fig. 1). The different proliferation rates of the urine cells and ADSCs in their xeno-free culture media were still evident after the electroporation of plasmids encoding the reprogramming genes (Fig. 1C and D). Therefore, the urine cells grew slower than ADSCs both before (Fig. 1A and B) and after (Fig. 1C and D) electroporation. Next, we wanted to confirm that the xeno-free culture media formulated in this study efficiently promoted the growth of the urine cells and ADSCs. To this end, we compared the growth of the cells in their corresponding xeno-free media with the non-xenofree media previously used by other groups, including DMEM/
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Fig. 1. Proliferation of human urine cells and ADSCs under xeno-free conditions. (A) The cells at p3 in each well of a 6-well plate were counted on days 1, 3, 5, and 7 after seeding; all wells were initially seeded with 5 104 cells/well. (B) The WST assay was performed to confirm the results shown in (A). (C) The urine cells and ADSCs were electroporated with three plasmids encoding reprogramming factors, and the cells were counted at several time points after electroporation. (D) The WST assay was performed to confirm the results shown in (C). (E) The proliferation rates of urine cells and ADSCs were examined under a variety of culture (xeno-free and non-xeno-free) conditions. (**P < 0.01).
Ham's F12 (1:1, v/v) containing 10% FBS and SingleQuot Kit CC-4217 REGM (for the urine cells) [21] and a-MEM containing 10% FBS (for the ADSCs) [23]. Our results demonstrated that the xeno-free media used in this study could indeed efficiently promote the growth of both the urine cells and ADSCs (Fig. 1E). In summary, both the urine cells and the ADSCs were cultured efficiently in their respective xeno-free media, although the urine cells showed a lower proliferative rate than the ADSCs. Thus, xenofree media would be suitable for the generation of xeno-free iPSCs.
total cell population between urine cells (~27%) and ADSCs (~13%) became obvious at passage 7 (Fig. 2A and B, middle panels). Strikingly, at passage 15, most of the urine cells (~98%) were annexin Vpositive, while only 17% of the ADSCs were annexin V-positive (Fig. 2A and B, bottom panels). These results indicated that the urine cells apparently senesce much earlier than the ADSCs in the xeno-free culture media used in this study. This result suggested that the use of low passage urine cells would be recommended for iPSC generation.
3.2. Cell death of urine cells and ADSCs at different passage numbers
3.3. Generation of xeno- and integration-free iPSCs from urine cells and ADSCs
We further examined whether the urine-derived cells and ADSCs could be maintained for an extended time period in their xeno-free media. The percentage of apoptotic (annexin V-positive) cells was comparably low (approximately 7e8% of the total cells) at low passages (~P3) for both cell types (Fig. 2A and B, top panels). However, the difference in the percentage of apoptotic cells in the
For therapeutic applications, the generation of strictly xeno- and integration-free iPSCs is essential. Therefore, we examined whether such xeno- and integration-free iPSCs could be generated by elaborately combining the EBNA1/OriP-plasmid transfection method [3] with the ECM(vitronectin)-based xeno-free hPSC culture conditions established in our laboratory [22].
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Fig. 2. The percentage of annexin V-positive cells was measured for the urine cells and ADSCs at passages 3, 7, and 15. The cells were stained with Hoechst 33342, Annexin V-FITC, and propidium iodide (PI). Hoechst 33342 stained the total cell population (left panels), while Annexin V stained the apoptotic and necrotic cells. Early apoptotic cells exclude PI (Annexin V-positive and PI-negative cells), whereas late-stage apoptotic and necrotic cells are positively stained for both Annexin V and PI (middle panels). Histograms (right panels) show the Annexin V-FITC intensity of the cell population. (A) Urine cells. (B) ADSCs.
First, the primary cells were electroporated with the three EBNA1/OriP-based plasmids encoding the 7 reprogramming factors (Oct4, Sox2, Lin28, L-Myc, Klf4, p53 shRNA, and SV40LT), and the resulting cells were then maintained under the ECM-based xenofree hPSC culture conditions until ESC-like colonies were formed. The ESC-like colonies were counted on days 18 and 23 after electroporation, and colonies were selected for expansion under the ECM-based xeno-free hPSC culture condition (Fig. 3A). Evidently, more colonies were formed from the ADSCs than the urine cells on days 18 and 23 after electroporation (~0.3% from the urine cells and ~0.4% from the ADSCs on day 23) (Fig. 3B). In summary, the xeno-free vitronectin-based hPSC culture condition was compatible with the episomal plasmid transfection method for the generation of xeno- and footprint-free iPSCs from both urine cells and ADSCs. Furthermore, the efficiency of iPSC generation via this method appeared to be higher than what was previously reported using the retroviral gene delivery/feeder-based culture system. 3.4. Gene expression profiles of the iPSCs derived from urine cells and ADSCs For detailed characterization, we chose two lines each from the iPSC lines that originated from the urine cells and the ADSCs (UiPSC1 and 2 and AiPSC1 and 2, respectively). First, we used DNA fingerprinting analysis to confirm that the iPSC clones we chose
were indeed derived from their original primary cells (either urine cells or ADSCs) (Supplementary Table 1). The iPSC lines derived from both cell types expressed multiple representative pluripotent cell markers, including Oct4, Sox2, SSEA4, Tra1-60, Tra1-81, Rex1, DNMT3B, and ZIC3, as detected by either immunostaining (Fig. 4A and B) or quantitative RT-PCR (Fig. 4C). Additionally, all the iPSC lines expressed a low level of differentiation markers for the three germ lineages, such as SOX1 and PAX6 (ectoderm), GATA2 and Brachyury (mesoderm), and AFP and SOX17 (endoderm) (Fig. 4D). Furthermore, genome-wide gene expression profiling analysis revealed a similar gene expression pattern between the iPSCs derived from urine cells and those derived from ADSCs (Fig. 5E). Scatter plot, heatmap, and hierarchical clustering of the genome-wide gene expression profiles all indicated a close correlation between UiPSC1, AiPSC1, and H9-hESC lines (Fig. 5AeG). PluriTest, a gene expression-based bioinformatic diagnostic test for evaluating pluripotency [24], showed that the iPSCs derived from urine cells and ADSCs were indeed fully reprogrammed pluripotent stem cells (Fig. 5H). 3.5. Characterization of the iPSCs derived from urine cells and ADSCs Bisulfide sequencing analysis revealed largely demethylated patterns in the Oct4 and Nanog promoters of the iPSC lines derived from both urine cells and ADSCs (Fig. 6A). On the contrary, the Oct4
Fig. 3. Generation of iPSCs from urine cells and ADSCs. (A) Schedule of the experiment. (B) ESC-like colonies were counted at day 18 and 23 post-electroporation. The colonies were immunostaining with an antibody targeting the undifferentiated cell marker Tra1-60. (**P < 0.01).
Fig. 4. Characterization of iPSCs generated from urine cells and ADSCs. The iPSC colonies that were generated and cultured for 23e25 passages were subjected to immunostaining (A, B) and real-time RT-PCR (C, D) to examine the undifferentiated cell markers Oct4, Sox2, SSEA4, Tra1-60, and Tra1-81. (A) Urine cell-derived iPSCs. (B) ADSC-derived iPSCs. A DIC image of a single colony is also shown. All the colonies expressed alkaline phosphatase, a marker for pluripotent stem cells. (C) Quantitative real-time RT-PCR was performed to detect the expression of the undifferentiated cell markers Oct4, nanog, Sox2, Rex1, DNMT3B, and Zic3. (D) Quantitative real-time RT-PCR was performed to detect the expression of the representative markers for the ectoderm (SOX1, PAX6), mesoderm (GATA6, Brachyury), and endoderm (AFP, SOX17) lineages. (**P < 0.01) Scale bar ¼ 100 mm.
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Fig. 5. Genome-wide gene expression profile analysis. (A-E) Scatter plot of gene expression profiles for iPSCs, hESCs, and primary cells. (F) Hierarchical clustering of the iPSCs, hESCs, and primary cells based on their gene expression patterns. (G) A heatmap of the microarray data from the iPSCs, hESCs, and primary cells. The genes shown in green are those that were upregulated, whereas the genes shown in red are those that were downregulated. (H) PluriTest analysis of the iPSCs, hESCs, and primary cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
and Nanog promoters of the primary cellsdboth urine cells and ADSCsdwere heavily methylated (Fig. 6A). These results were consistent with the iPSC-specific expression of the Oct4 and Nanog genes. The iPSCs retained pluripotency because these cells were able to differentiate into cells from all three germ layers (Fig. 6B and C). Gbanding chromosomal analysis indicated that there were no gross chromosomal aberrations in the iPSC lines generated and cultured in the ECM-based xeno-free hPSC medium (Supplementary Fig. 2A). Importantly, PCR analysis using episomal plasmid-specific primer pairs clearly demonstrated that the episomal plasmids were not integrated into the host chromosomes (Supplementary Fig. 2B). Taken together, our results reported that xeno- and footprintfree iPSCs were efficiently generated from urine cells, which can
be obtained in a non-invasive manner. Additionally, we showed that our combined method worked with other primary cells, such as ADSCs, which can also be easily acquired from patients. Therefore, our ECM-based (feeder-independent) method of generating xeno- and footprint-free iPSCs established in this study would be of great clinical value by providing an unlimited number of autologous or immune-compatible cell sources for future iPSC-based cell replacement therapy. 4. Discussion Clinically applicable, safe iPSCs should ideally be generated using chromosome integration-free methods under xeno-free culture conditions. Until now, several methods for producing integrationfree iPSCs have been developed; among these, the EBNA1/OriPbased plasmid method [1e3], the mRNA transfection method [4],
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Fig. 6. DNA methylation of Oct4 and nanog promoters and pluripotency analysis in vitro and in vivo. (A) Bisulfite sequencing analysis showed that the Oct4 and nanog promoters in the iPSCs were largely demethylated, similar to what was observed in hESCs. (B) In vitro pluripotency test. The iPSCs spontaneously differentiated, and the expression levels of representative markers of the ectoderm (nestin, beta-III tubulin), mesoderm (smooth muscle actin, PECAM), and endoderm (a-fetoprotein, FoxA2) lineages were examined. (C) The derivatives of the three germ layers were detected in teratomas, which formed approximately 9 weeks after iPSC administration (2 106 cells) into NOD/SCID mice. Scale bar ¼ 100 mm.
and the Sendai viral method [5] are commonly used. Therefore, an ECM-based xeno-free hPSC culture condition that supports the efficient generation of iPSCs in combination with the aforementioned integration-free methods is greatly in demand. For the purpose of iPSC-based cell therapy, we sought to generate xeno- and integration-free iPSCs using the ECM-based (feeder-independent) xeno-free hPSC culture medium we established. As an ECM, we used human vitronectin that was purified from human plasma, and the xeno-free medium was formulated around a protein kinase C inhibitor, which is critical for the maintenance of stemness [22]. Although this xeno-free hPSC culture
system was shown to support the efficient generation of iPSCs using the retrovirus-mediated method, its compatibility with an integration-free method for the generation of iPSCs had not been tested. This is a very important issue to explore for future cell replacement therapeutic approaches. Therefore, in this study, we tested whether integration- and xeno-free iPSCs were generated efficiently by elaborately combining the EBNA1/OriP-based plasmid method and the ECM-based xeno-free hPSC culture medium. The choice of cell sources for iPSC generation is also an important factor to consider. Ideal cell sources are cells that can be obtained from patients with no or minimal invasiveness and can also
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be reprogrammed into iPSCs with high efficiency. In this study, we tested whether xeno- and integration-free iPSCs can be easily generated from urine cells. Urine cells are a heterogeneous population of cells comprising renal tube epithelial cells, urothelial cells, fibroblast-like cells, and urine-derived adult stem cells (i.e., mesenchymal stem cells) [25]. To generate strictly xeno-free iPSCs, we grew the primary cells, urine cells and ADSCs, under xeno-free conditions starting from biopsy. Although a xeno-free primary cell culture medium for ADSCs was commercially available, no xeno-free medium was available for the urine cells. Therefore, we formulated a new xenofree culture medium for these cells by replacing bovine serum albumin with human serum albumin from REGM, and we mixed the modified REGM with MesenGro® medium at a 1:1 ratio (v/v). The urine cells and the ADSCs grew well in their respective xeno-free media; however, the ADSCs grew faster than the urine cells. Intriguingly, the proportion of dead cells among the total cells was low (7e8%) at early passages (P3) in both cell types, although the urine cells tended to die much faster than the ADSCs as the passage number increased (27% vs. 13% at P7 and 98% vs. 17% at P15) (Fig. 2). It is currently not clear whether this result was caused by an intrinsic characteristic of the urine-derived cells and ADSCs or by the xeno-free culture conditions used for their culture. We believe that the former possibility is more likely because the urine cells tended to enter senescence much earlier than the ADSCs, regardless of which media were used for culture (data not shown); however, this would not affect the generation of iPSCs because we mostly use cells with low passage numbers (less than 4) for iPSC generation. The results of this study showed that the number of iPSCs generated from the adipose-derived stromal cells was higher than the number derived from the urine cells (0.38% vs. 0.29% at day 23). The efficiency of ADSC-derived iPSC generation using our integration- and xeno-free method appeared to be higher than that reported in a previous paper describing the generation of AiPSCs using retroviral mediated gene delivery (0.2% on MEFs and 0.01e0.03% on Matrigel) [18]. The efficiency of UiPSC generation was within the range previously reported for UiPSC generation using the retroviral method on MEF feeder cells (0.01e4%) [21]. It was generally thought that feeder-free culture conditions were less efficient for iPSC generation than the feeder-dependent culture system [18]. In this regard, the apparently high efficiency of iPSC generation in the feeder-free conditions in our study may reflect the intrinsic enhancing activity of our ECM-based xeno-free hPSC culture system, which was previously described to be significantly higher than the conventional MEF-dependent method [22]. The efficiency of iPSC generation may also be affected by the proliferation rate of the primary cells that are being used. Although there are reports arguing that a negative correlation exists between the growth rate and the iPSC formation efficiency [26], this issue remains unclear due to multiple contradictory reports [27,28]. At present, it is not clear whether the more efficient iPSC formation of ADSCs compared to urine cells is caused by the higher proliferative activity or any intrinsic property of ADSCs. In this study, we have demonstrated two important results regarding the generation of xeno- and integration-free iPSCs: (1) Our ECM-based xeno-free hPSC culture system supported the efficient generation of integration-free iPSCs from both urine cells and ADSCs, indicating that the xeno-free hPSC culture system functions well with the integration-free iPSC generation method. (2) Early passage urine cells are good cell sources for iPSC generation due to the non-invasive nature of collection and their high reprogramming efficacy. Taken together, this study reported the efficient generation of xeno-free and footprint-free iPSCs from urine cells, which can be obtained non-invasively, and also provides an efficient and
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convenient platform for the generation of therapeutically applicable iPSCs for cell therapy. 5. Conclusions In this study, we reports the efficient ECM-based (feeder-independent) generation of integration- and xeno-free iPSCs from easily obtainable urine cells and hence, provides a platform that can be used to generate iPSCs for cell replacement therapy in the foreseeable future. Author contributions D.-Y. H. designed the project and wrote the manuscript. K.-I. L and H.-T. K. equally contributed by performed most experiments. Acknowledgments This work was supported by grants from the Stem Cell Research Program (2010-0020350) and 2012M3A9C7050130 from the MSIP, and A120254-1201-0000200 from the Ministry of Health & Welfare, Korea. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.05.059. References [1] Jia F, Wilson KD, Sun N, Gupta DM, Huang M, Li Z, et al. A nonviral minicircle vector for deriving human iPS cells. Nat Meth 2010;7:197e9. [2] Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin II , et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science (New York, Ny) 2009;324:797e801. [3] 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 Meth 2011;8:409e12. [4] Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F, et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 2010;7:618e30. [5] Fusaki N, Ban H, Nishiyama A, Saeki K, Hasegawa M. Efficient induction of transgene-free human pluripotent stem cells using a vector based on sendai virus, an RNA virus that does not integrate into the host genome. proceedings of the Japan academy series B. Phys Biol Sci 2009;85:348e62. [6] Martin MJ, Muotri A, Gage F, Varki A. Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat Med 2005;11:228e32. [7] Heiskanen A, Satomaa T, Tiitinen S, Laitinen A, Mannelin S, Impola U, et al. Nglycolylneuraminic acid xenoantigen contamination of human embryonic and mesenchymal stem cells is substantially reversible. Stem Cells (Dayt Ohio) 2007;25:197e202. [8] Hu W, Yan Q, Fang Y, Qiu Z, Zhang S. Generation of human induced pluripotent stem cells with non-integrating episomal vectors and xeno-free culture system. J Anim Veterinary Adv 2013;12:1114e22. [9] Nakagawa M, Taniguchi Y, Senda S, Takizawa N, Ichisaka T, Asano K, et al. A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells. Sci Rep 2014;4:3594. [10] Fukusumi H, Shofuda T, Kanematsu D, Yamamoto A, Suemizu H, Nakamura M, et al. Feeder-free generation and long-term culture of human induced pluripotent stem cells using pericellular matrix of decidua derived mesenchymal cells. PloS One 2013;8:e55226. [11] 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;131:861e72. [12] Takenaka C, Nishishita N, Takada N, Jakt LM, Kawamata S. Effective generation of iPS cells from CD34þ cord blood cells by inhibition of p53. Exp Hematol 2010;38:154e62. [13] Haase A, Olmer R, Schwanke K, Wunderlich S, Merkert S, Hess C, et al. Generation of induced pluripotent stem cells from human cord blood. Cell Stem Cell 2009;5:434e41. [14] Giorgetti A, Montserrat N, Aasen T, Gonzalez F, Rodriguez-Piza I, Vassena R, et al. Generation of induced pluripotent stem cells from human cord blood using OCT4 and SOX2. Cell Stem Cell 2009;5:353e7. [15] Loh YH, Hartung O, Li H, Guo C, Sahalie JM, Manos PD, et al. Reprogramming of T cells from human peripheral blood. Cell Stem Cell 2010;7:15e9.
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