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Induced Pluripotent Stem Cell-Derived Models for mtDNA Diseases
CHAPTER NINETEEN
Induced Pluripotent Stem Cell-Derived Models for mtDNA Diseases Riikka H. Hämäläinen1 Research Programs Unit, Molecular Neurology, Biomedicum-Helsinki, University of Helsinki, Helsinki, Finland 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Induced Pluripotent Stem Cells 2.1 iPSC-derived disease models 3. Mitochondrial Disease 3.1 iPSCs from patients with mtDNA mutations 3.2 Utilization of iPSCs as mtDNA disease models 4. Conclusion 5. Generation of iPSCs from mtDNA Disease Patients 5.1 Materials 5.2 Protocol 5.3 Notes Acknowledgment References
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Abstract Mitochondrial disease due to mutations in the mitochondrial DNA (mtDNA) is a common cause of human inherited disorders. Targeted modification of the mitochondrial genome has not succeeded with the current transgenic technologies. Furthermore, readily available cultured patient cells often do not manifest the disease phenotype. Therefore, pathogenic mechanisms underlying these disorders remain largely unknown, as the lack of model systems has hampered mechanistic studies. Stem cell technology has opened up new ways to use patient cells in research, through generation of induced pluripotent stem cells (iPSCs) and differentiation of these to diseaserelevant cell types, including, for example, human neurons and cardiomyocytes. Here, we discuss the use of iPSC-derived models for disorders with mtDNA mutations.
Methods in Enzymology, Volume 547 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-801415-8.00019-9
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2014 Elsevier Inc. All rights reserved.
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1. INTRODUCTION Mitochondrial diseases are clinically heterogeneous group of disorders that may affect only a single organ, but often involve multiple organ systems (Nunnari & Suomalainen, 2012), most typically, the patients present with prominent neurologic, cardiomyopathic, or myopathic features. Mitochondrial disease may arise from a defect in either the nuclear or mitochondrial genome, and thus, the mode of inheritance may be recessive, dominant, X-linked, or maternal. Patients with mitochondrial DNA (mtDNA) mutations show exceptional clinical variability, but the mechanistic basis of the spectrum of disorders is unknown: introduction of mtDNA mutations into mitochondria by transgenic technologies has not been successful, and thus only a few mouse models that have been generated through cybrids are available for pathophysiological studies (Inoue et al., 2000; Lin et al., 2012; Nakada & Hayashi, 2011). Cultured cells from patients are available for research purposes; however, the disorders are often highly tissue specific and, based on our own experience, only less than half of the disorders manifest a pathological phenotype in cultured cells. Generation of induced pluripotent stem cells (iPSCs) has revolutionized the field of disease modeling in recent years (Takahashi & Yamanaka, 2006; Takahashi et al., 2007). Now, iPSCs can routinely be generated from samples that are readily available from patients, e.g., skin biopsies or blood samples. The iPSCs can then further be differentiated toward the desired target cell populations, which can then be used as research material both in mechanistic studies and in treatment trials. Major advances have already been made, for example, in neurodegenerative disease and cardiac disease fields (Matsa, Burridge, & Wu, 2014; Peitz, Jungverdorben, & Brustle, 2013). We discuss here the use of iPSC-based disease modeling for patients with mtDNA mutations and mitochondrial disease.
2. INDUCED PLURIPOTENT STEM CELLS Embryonic stem (ES) cells have two unique properties: (1) they are able to self-renew and divide indefinitely, and (2) they are pluripotent and thus can differentiate to any cell type of an adult organism. iPSCs are somatic cells, e.g., adult fibroblasts, which have been driven backward in differentiation to an ES cell-like state (Takahashi & Yamanaka, 2006; Takahashi et al., 2007). iPSCs possess the two unique properties of ES cells,
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and thus they can be both propagated in culture indefinitely as iPSCs and differentiated basically to any adult cell type, such as neurons or cardiac cells, through in vitro differentiation. These two ES cell-like properties make iPSCs highly useful for different research and therapeutic purposes. Reprogramming of somatic cells to a pluripotent state was initially achieved by ectopic expression of four transcription factors highly expressed in ES cells, namely, Oct4, Sox2, Klf4, and c-Myc (Takahashi & Yamanaka, 2006; Takahashi et al., 2007). Since the original reports, different variations of the transgene cocktail, for example, omitting the c-Myc, which has oncogenic properties, have been reported to result in fully reprogrammed iPSCs (Nakagawa et al., 2008); however, the initial so-called Yamanaka-cocktail is still widely used because of its efficiency. In the original reports, the transgenes were introduced into the cells by retroviral infection (Takahashi & Yamanaka, 2006; Takahashi et al., 2007). With evolving technology, novel methods to introduce the reprogramming transgenes to host cells have been reported, including nonintegrative methods like Sendai virus or episomal plasmid vectors (Ban et al., 2011; Okita et al., 2011). Further, reprogramming of human cells to pluripotency has also been achieved by DNA-free methods using direct delivery of either mRNAs or proteins (Kim et al., 2009; Warren et al., 2010; Yoshioka et al., 2013). In addition to fibroblasts, iPSCs have been now generated from a variety of different cell types including blood cells and epithelial cells from urine samples (Staerk et al., 2010; Zhou et al., 2012).
2.1. iPSC-derived disease models One of the research fields most enhanced by iPS technology is disease modeling. iPSCs have already been generated from a variety of patients with different diseases and the list keeps growing (Park et al., 2008; Peitz et al., 2013). While animal models have been crucial for mechanistic studies of human disease pathology, models that thoroughly reproduce human pathology are rare and, for example, drug trials done in animal models do not always work in human patients (Inoue & Yamanaka, 2011). Challenges exist also in iPSC-based disease modeling. While generation of iPSCs is fairly robust, differentiation of the iPSCs to the desired cell type is not trivial and achieving pure target cell populations is at least difficult if not impossible. Also, a genetic defect in a cell may not always lead to functional manifestations, and different stressors may be needed to challenge the
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cultures and to mimic the physiological disease niches. Further, many lateonset diseases may not easily manifest their key features in short-term cultures. However, different stressors or nutrient deprivation may be used to enhance the aging process in the in vitro culture system. A good control setting is critical for all model systems, and this becomes even more evident with iPSC-based modeling, as, in addition to the normal biological variation between individuals, the variation between different iPSC clones may be remarkable. The current standard in the field is to use nondisease control lines together with gene-edited isogenic control lines (Soldner et al., 2011). While the isogenic control lines reduce noise significantly, multiple clones will still need to be analyzed to avoid off-target effects and clonal variation between different lines. Despite the challenges, iPSC-based disease modeling has already proven to be an efficient tool to study disease mechanisms and remarkable advances have been achieved. Successful model lines, which recapitulate manifestations seen in patient tissue, have been generated for various diseases. For example, underlying mechanisms and potential targets for therapy have been identified for severe human neurodegenerative disorders like amyotrophic lateral sclerosis and Alzheimer’s disease (Donnelly et al., 2013; Kondo et al., 2013) and successful drug tests have been done in iPSC-derived cardiac cells from patients with long QT syndrome (Lahti et al., 2012; Terrenoire et al., 2013).
3. MITOCHONDRIAL DISEASE Mitochondria are best known for their function in transforming the energy of nutrients in the form of ATP, through cellular oxidative phosphorylation. In addition to ATP production, mitochondria take part in several other cellular metabolic functions, and their signals affect whole cell signaling, including redox-dependent nutrient sensor and ROS signaling, cellular proliferation, differentiation, and programmed cell death. Mitochondria are under a dual genetic control. They possess multiple copies of their own DNA (mtDNA), which encodes 13 subunits of the mitochondrial respiratory chain (RC) enzymes as well as the mitochondrial tRNAs and rRNAs required for the synthesis of these proteins. All other mitochondrial proteins are encoded by the nuclear genome. Despite the small size of the mitochondrial genome, mtDNA mutations underlie a vast number of human inherited disorders. In western populations, mtDNA mutations cause a disease in every 1:5000 individuals
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(Skladal, Halliday, & Thorburn, 2003), and every 1:200 newborns carries a potentially pathogenic mtDNA mutation (Elliott, Samuels, Eden, Relton, & Chinnery, 2008). Over 200 mtDNA point mutations have to date been reported to underlie human disease (Ylikallio & Suomalainen, 2012). Clinical features of these diseases vary largely from infantile multisystem disorders to adult-onset myopathies and neurodegeneration, and in fact, mitochondrial disease can occur in any organ system with any age of onset (Ylikallio & Suomalainen, 2012). The manifestations may vary even in patients carrying exactly the same mutations: for example, m.3243A>G that leads to diabetes and deafness in some patients may lead to a brain phenotype and strokes or cardiac disease in others (Chae et al., 2004; Goto, Nonaka, & Horai, 1990; van den Ouweland et al., 1992). A typical feature of a pathogenic mtDNA mutation is heteroplasmy; the patient cells carry both normal and mutant mtDNA. The disease manifests when the mutant mtDNA amount exceeds a threshold, which may vary between tissues and different individuals (Ylikallio & Suomalainen, 2012). While segregation of mutated mitochondria and tissue-specific threshold levels are important factors in determining the clinical outcome of mtDNA mutations, other, as yet unidentified, factors also play an important role. This is evident, as even homoplasmic mtDNA mutations may cause variable clinical phenotypes (Gropman et al., 2004). To identify these additional factors and to study mechanisms underlying these disorders, good model systems are needed.
3.1. iPSCs from patients with mtDNA mutations Since other model systems for mtDNA disorders are scarce, high hopes are set for the use of iPSCs in studying these diseases. Initial concerns of whether cells that have a defect in ATP production were able to reprogram properly have been shown to be unwarranted, and iPSCs have been generated from patients with different mtDNA mutation types, both point mutations (Folmes et al., 2013; Fujikura et al., 2012; Ha¨ma¨la¨inen et al., 2013) and large mtDNA deletions (Cherry et al., 2013; Table 19.1). Independent of the mutation type or frequency, all reported iPSCs with mtDNA mutations have shown normal ES cell-like morphology and high expression of pluripotency marker genes. All reported lines have also been able to differentiate toward all three germ layers (Cherry et al., 2013; Folmes et al., 2013; Fujikura et al., 2012; Ha¨ma¨la¨inen et al., 2013). However, while cells harboring point mutations in either tRNA or RC protein-coding genes have
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Table 19.1 Published iPSCs with mtDNA mutations Cell types differentiated Disease Mutation from iPSCs References
Diabetes (MIDD)
A3243G
–
Fujikura et al. (2012)
MIDD/MELAS A3243G
Neurons
Ha¨ma¨la¨inen et al. (2013)
MELAS
G13513A
–
Folmes et al. (2013)
Pearson syndrome
10949_13449del Hematopoietic cells
Cherry et al. (2013)
MIDD, maternally inherited diabetes and deafness; MELAS, mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes.
shown normal efficiency and kinetics of reprogramming, the patient cells with large mtDNA deletions showed low reprogramming efficiency and the process was significantly slower than in control cells (Cherry et al., 2013). In all the studies reported so far, mtDNA heteroplasmy has shown specific segregation patterns, both during reprogramming and the subsequent culture and differentiation of the iPSCs. During reprogramming, all mtDNA mutations segregated in a bimodal fashion leading to iPS clones with either no detectable mutant mtDNA, or those with over 50% of the total mtDNA pool in a cell being mutated (Cherry et al., 2013; Folmes et al., 2013; Fujikura et al., 2012; Ha¨ma¨la¨inen et al., 2013). During culture of the iPSCs, the tRNA mutation (A3243G in tRNA-LeuUUR) remained with stable mutant mtDNA amount, whereas the protein-coding RC mutation (G13513A in ND5) frequency decreased. The large mtDNA deletion (10949_13449del affecting five genes; ND4, ND5, tRNA-LeuCUN, tRNA-SerAGY, and tRNA-His) was stable in certain iPSC clones, but decreased in others until undetectable. On the other hand, after reprogramming, during differentiation of the iPSCs, all mtDNA mutant cell lines retained stable heteroplasmy level: no significant changes in the heteroplasmy levels were found between the starting iPSCs and the differentiated cells in any of the studies (Folmes et al., 2013; Fujikura et al., 2012; Ha¨ma¨la¨inen et al., 2013). These segregation patterns of the mutated mtDNA fit well with data published previously from animal studies. The bimodal segregation of the mutations during reprogramming suggests a mitochondrial bottleneck, similar to the one seen in germ cells and during epiblast specification (Cao et al., 2007; Lee et al., 2012). Furthermore, purifying
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selection of mutations affecting protein-coding genes has been reported in mouse germ cells (Stewart et al., 2008). This seems to take place also in stem cells and fits well with the fact that the ND5 mutation frequency reduced during iPSC culture, while the tRNA-LeuUUR stayed stable (Folmes et al., 2013; Ha¨ma¨la¨inen et al., 2013). Due to the bimodal segregation of the mutations during reprogramming, it is possible to derive clones from one individual with both high mtDNA mutation frequency and no detectable mtDNA mutations. This is a major advantage, since it enables simultaneous generation of both disease model lines and their isogenic control lines, i.e., same nuclear background, from the same patients without additional gene editing. All the lines with mtDNA mutations reported so far have shown normal differentiation potential, and all heteroplasmic mtDNA variants have been stable during differentiation, allowing generation of desired target cell populations with high and low mutations frequencies. In addition to proving that generation of iPSC lines and their differentiation to the desired target cell types with high mtDNA mutation levels is feasible for different mtDNA mutation types, the initial studies have also proven that iPSC-derived cells manifest disease phenotypes typical for patient tissues, and that the manifestations are cell type specific. Cherry et al. showed that fibroblasts derived from bone marrow of a Pearson patient manifested a combined RC defect. iPSCs derived from these fibroblasts grew slowly, and this may at least partially explain the slow appearance of colonies during reprogramming of these cells. Also, the Pearson iPSCs with large mtDNA deletion manifested an RC deficiency. The cells showed low mitochondrial membrane potential, low cellular respiration, and partial compensation of this with increased glycolysis. However, differentiation of hematopoietic cells was not significantly altered between cells with different levels of mutant mtDNA. Among the in vitro differentiated erythroblasts, a high level of sideroblasts (abnormal erythroblasts with pathologic iron granule depositions in mitochondria) were seen specifically in the cultures with high frequency of deletion (Cherry et al., 2013), which is consistent with sideroplastic anemia being a hallmark of Pearson syndrome (Pearson et al., 1979). We showed in our study that while the fibroblasts with G3243A mutation in mtDNA manifested a mild combined RC defect, no RC defect existed in the iPSCs. The iPSCs with this tRNA-LeuUUR mutation grew normally and also the reprogramming kinetics was similar to control cells (Ha¨ma¨la¨inen et al., 2013). Further, we showed that in vitro differentiated neurons with high mtDNA mutation frequency
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manifested an isolated Complex I deficiency, which is typical also for patient tissues (Ciafaloni et al., 1992; Goto et al., 1992). Our data clearly show that manifestation of the RC defect is dependent on the cellular context, and stresses the importance of studying the relevant cell types for the specific diseases.
3.2. Utilization of iPSCs as mtDNA disease models iPSCs provide a powerful tool for studying mitochondrial disease, since they can be differentiated toward basically any adult cell type. Apart from neuronal and hematopoietic cells which have currently been generated from iPSCs with mtDNA mutations (Cherry et al., 2013; Ha¨ma¨la¨inen et al., 2013), it is also possible to derive many other cell types that are relevant for mtDNA disease from these same cells. Directed differentiation of iPSCs toward, for example, cardiac cells (Lahti et al., 2012; Terrenoire et al., 2013), retinal pigment epithelia (Kamao et al., 2014; Krohne et al., 2012), hepatic cells (Si-Tayeb et al., 2010; Sullivan et al., 2010), and pancreatic β-cells (Shahjalal et al., 2014) has already been successfully achieved. Apart from generating desired target cell types through directed differentiation protocols, iPSCs can be differentiated through teratoma formation. This does not allow for controlling the outcome of the differentiation, but will generate highly differentiated cells and tissues. The teratomas can be used to assess RC function in several different tissue types (Castro et al., 2010; Ha¨ma¨la¨inen et al., 2013). The bimodal segregation of mutant and wild-type mtDNA upon reprogramming resembles mtDNA bottleneck upon epiblast specification (Ha¨ma¨la¨inen et al., 2013; Lee et al., 2012), and this allows for using iPSCs and reprogramming to study mitochondrial bottleneck and the mechanisms that control mtDNA segregation during early embryonic development. As cell culture conditions still differ from mature tissues, some mitochondrial disorders do not manifest disease phenotype even when differentiated into neurons or cardiomyocytes, but the manifestation would require differentiation within a mature tissue. Obtaining good-quality iPSCs is a timeconsuming, laborious, and expensive process, and—similar to mouse models—the knowledge of whether the model is actually useful will only be available after iPSC generation and subsequent differentiation have been performed. Our experience for mtDNA mutations, however, has been promising as relevant, potentially pathologic physiological pathways mimicking patient tissues have been produced in culture.
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4. CONCLUSION iPSC-based disease modeling has provided a powerful tool for generating human patient-derived postmitotic cells, and especially so for heteroplasmic mtDNA mutations, which otherwise lack proper disease models (Fig. 19.1). iPSC-derived materials have already been successfully used for both mechanistic studies and treatment trials. It is clear that iPSC line generation from cells with mtDNA mutations is feasible. Furthermore, the bimodal segregation of mutant mtDNA upon reprogramming results in simultaneous generation of iPSC clones with high mutation frequencies as well as isogenic control lines with no detectable mutations. The heteroplasmy remains stable after reprogramming, enabling derivation of desired target cells, such as neurons or cardiomyocytes, with high or low mutation levels. The initial studies have also shown that iPSC-derived cells, hematopoietic cells from a Pearson patient, as well as neurons from an MELAS
Figure 19.1 iPSC-based disease modeling for disorders with mtDNA mutations. Fibroblasts (or other cell types) from patients can be reprogrammed to an induced pluripotent state. During reprogramming, heteroplasmic mtDNA mutations segregate giving rise to cells with either high or low mutation frequency. These cells can then be used as both disease models and isogenic controls for the disease lines. The cells can further be differentiated to any desired target cell type where tissue-specific disease manifestations can be studied. This provides a powerful tool to study disease mechanisms and to test potential therapeutic agents.
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patient manifest symptoms typically seen in patient tissues. These data suggest that iPSCs will provide a powerful tool to studying disorders with heteroplasmic mtDNA mutations.
5. GENERATION OF iPSCs FROM mtDNA DISEASE PATIENTS Generation of iPSCs from fibroblasts can be achieved by several different methods. We have used both retroviral (Takahashi et al., 2007) and episomal (Okita et al., 2011) methods and describe here the episomal plasmid method originally published by Okita and Yamanaka (Okita et al., 2013).
5.1. Materials Patient fibroblasts from skin biopsy. Episomal plasmids from Addgene: 27077 (pCXLE-hOCT3/4-shp53-F), 27078 (pCXLE-hSK), and 27079 (pCXLE-hMLN). Mouse embryonic fibroblast (MEF) feeder cells. Prepared from 12.5dpc ICR embryos, mitotically arrested with mitomycin-c treatment. Fibroblast culture media: DMEM (high glucose), glutamax, penicillin/ streptomycin, supplemented with 10% FBS. hES media: KO-DMEM, nonessential amino acids, glutamax, betamercaptoethanol (0.1 mM), and penicillin/streptomycin, supplemented with 20% KOSR (Life Technologies, Grand Island, NY, USA) and 10 ng/ml bFGF: used during iPSC establishment Essential eight medium (Life technologies): used during iPSC culture. Uridine Phosphate-buffered saline (PBS), Mg2+, and Ca2+ free TrypLE Select (Life Technologies) EDTA 0.5 mM Gelatin 0.1% Matrigel (BD Biosciences, Bedford, MA, USA) Trypan blue Freezing media (E8 + 10% DMSO) Hematocytometer or automated cell counter Electroporator e.g., Neon Transfection system (Life Technologies, Carlsbad, CA, USA) 15-ml centrifuge tube 6-cm culture dishes
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6-well culture dishes 4-well culture dishes scalpels cryovials
5.2. Protocol 1. Expand low passage fibroblasts (see Notes 1 and 2) in an appropriately sized dish in fibroblast medium. One 6-cm dish can yield about 1–1.5 106 cells. Once the desired cell number is reached, proceed to electroporation. 2. Prepare one 6-cm plate per cell line. Apply gelatin (0.1%) to the surface of the dish, aspirate, and allow to dry uncovered in the biosafety cabinet. Add fibroblast media (see Note 3) and put the plates in 37 C; 5% CO2. 3. When the fibroblasts reach 70–80% confluency (see Note 4), harvest them using enzymatic dissociation with TrypLE (5 min at 37 C), collect by centrifugation at 750 rpm for 3 min, resuspend in fresh medium, and count viable cells using trypan blue exclusion and hematocytometer or automated cell counter. 4. Collect 700,000 cells, wash with PBS, and resuspend in 120 μl of buffer R. 5. Mix 1 μg of each of the three plasmids pCXLE-hOCT3/4-shp53-F (27077), pCXLE-hSK (27078), and pCXLE-hMLN (27079) (all from Addgene) with the cells. Electroporate according to the system instructions (see Note 5) with 1650 V, 10 mS, and three pulses. Transfer the cells directly from the tip to the freshly prepared 6-cm dishes. Culture overnight in 37 C; 5% CO2. This is considered as day 1. 6. On days 2 and 4 feed the cells with fresh fibroblast media. 7. On day 5 prepare feeder plates for passaging the fibroblasts. Apply gelatin (0.1%) to the surface of a 6-well dish, aspirate, and allow to dry uncovered in the biosafety cabinet. Plate 100,000 mitotically inactivated MEFs per well in fibroblast media and let the cells adhere overnight in 37 C; 5% CO2. 8. On day 6 split the cells to the feeder cells. Change fresh media to the feeder cell plates. Harvest electroporated fibroblasts using TrypLE (5 min at 37 C), collect by centrifugation at 750 rpm for 3 min, resuspend in fresh fibroblast medium, and count viable cells. Plate 50,000–100,000 cells per well (see Note 6) on the feeder cells.
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9. On day 7 change to hES media and feed every second day with fresh media. Monitor the wells for appearance of characteristic iPSC growth foci which typically become visible after 2 weeks in culture (Fig. 19.2). 10. Colonies suitable for picking may form as early as day 20; however, clones will typically continue to arise until days 28–35. 11. Pick colonies mechanically, dividing large colonies into small pieces with a scalpel, a pulled glass pipette, or 30-gauge needle. Plate the picked colonies on freshly prepared 4-well dishes (see Note 7) with feeder cells and hES media. 12. A few initial passages should be done mechanically and the cells kept on feeder cells with hES media (see Note 8). Typical colony types are presented in Fig. 19.2. 13. After establishing the lines, the cells can be transferred to Matrigelcoated plates and cultured in E8 media using EDTA for passaging. Coat the plates with Matrigel and equilibrate to RT (1–2 h). Prewarm E8 medium at RT. 14. Aspirate the medium and rinse the cells with PBS. Add 0.5 mM EDTA and incubate at RT for 5–8 min. Proceed when the cells start to separate and round up. Aspirate the EDTA solution and add E8 to the dish. Remove the cells from the well by gently squirting medium and pipetting the colonies up but do not scrape the cells from the dish. Collect cells in a 15-ml conical tube. Add E8 to a matrigel-coated plate and plate the cells in a desired ratio. 15. Grow the cells in 37 C, 5% CO2 incubator and feed daily or every second day. Passage when needed. 16. Once the iPSC lines have been established, they can be frozen down. Prepare freezing media by adding 10% DMSO to E8 media. Place on ice until use.
Figure 19.2 Typical colony types present in early passage iPSC lines. (A) A perfect colony with clearly defined edges and high cell density. (B) A moderate quality colony with lower cell density. (C) A differentiating colony. The dense part with defined borders can be cut out and cultured further. (D) Differentiated cells.
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17. Treat the cells with EDTA as when splitting, but instead of media add 1 ml of ice-cold freezing media to each well. Remove the cells by gently squirting the colonies from the well. Collect the cells in cryovials, resuspend gently, and place on ice. Transfer first to 80 C and after 24 h to 150 C or liquid nitrogen for longer storage.
5.3. Notes 1. To avoid selecting against mtDNA mutations, it is necessary to include uridine (200 μM) and pyruvate to the culture media during all stages of cell culture (King & Attardi, 1989). 2. A high growth rate of the starting fibroblasts is critical for good reprogramming efficiency. 3. Antibiotics are optional in all culture media; however, immediately after electroporation, it is best to culture the cells overnight without antibiotics, to allow them to recover from the electroporation without any additional stress. 4. The cells should be in an exponential growth phase. 5. For electroporation we use a Neon electroporation system (Invitrogen), but other systems should work as well. The method is explained for Neon transfection system with 100 μl tips, but also 10 μl tips can be used. 6. The plating density depends on the growth rate of the fibroblasts. If the cells grow very fast, lower densities may be useful to prevent overgrowth of the cells during subsequent culture. If the plates become too dense, this will hinder the proper formation of the iPSC colonies and the fibroblasts may detach from the plates. 7. Even though the surface size of a 24-well dish is the same as a 4-well dish, we find it easier to use 4-well dishes, as these have lower edges, which will make the mechanical cutting easier. 8. During initial passaging, some differentiation and cell death may occur. This is normal, and mechanical cutting will help to select for the good pluripotent cells.
ACKNOWLEDGMENT Anu Suomalainen-Wartiovaara is thanked for valuable comments to the manuscript.
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Induced Pluripotent Stem Cell-Derived Models for mtDNA Diseases Riikka H. Hämäläinen1 Research Programs Unit, Molecular Neurology, Biomedicum-Helsinki, University of Helsinki, Helsinki, Finland 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Induced Pluripotent Stem Cells 2.1 iPSC-derived disease models 3. Mitochondrial Disease 3.1 iPSCs from patients with mtDNA mutations 3.2 Utilization of iPSCs as mtDNA disease models 4. Conclusion 5. Generation of iPSCs from mtDNA Disease Patients 5.1 Materials 5.2 Protocol 5.3 Notes Acknowledgment References
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Abstract Mitochondrial disease due to mutations in the mitochondrial DNA (mtDNA) is a common cause of human inherited disorders. Targeted modification of the mitochondrial genome has not succeeded with the current transgenic technologies. Furthermore, readily available cultured patient cells often do not manifest the disease phenotype. Therefore, pathogenic mechanisms underlying these disorders remain largely unknown, as the lack of model systems has hampered mechanistic studies. Stem cell technology has opened up new ways to use patient cells in research, through generation of induced pluripotent stem cells (iPSCs) and differentiation of these to diseaserelevant cell types, including, for example, human neurons and cardiomyocytes. Here, we discuss the use of iPSC-derived models for disorders with mtDNA mutations.
Methods in Enzymology, Volume 547 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-801415-8.00019-9
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1. INTRODUCTION Mitochondrial diseases are clinically heterogeneous group of disorders that may affect only a single organ, but often involve multiple organ systems (Nunnari & Suomalainen, 2012), most typically, the patients present with prominent neurologic, cardiomyopathic, or myopathic features. Mitochondrial disease may arise from a defect in either the nuclear or mitochondrial genome, and thus, the mode of inheritance may be recessive, dominant, X-linked, or maternal. Patients with mitochondrial DNA (mtDNA) mutations show exceptional clinical variability, but the mechanistic basis of the spectrum of disorders is unknown: introduction of mtDNA mutations into mitochondria by transgenic technologies has not been successful, and thus only a few mouse models that have been generated through cybrids are available for pathophysiological studies (Inoue et al., 2000; Lin et al., 2012; Nakada & Hayashi, 2011). Cultured cells from patients are available for research purposes; however, the disorders are often highly tissue specific and, based on our own experience, only less than half of the disorders manifest a pathological phenotype in cultured cells. Generation of induced pluripotent stem cells (iPSCs) has revolutionized the field of disease modeling in recent years (Takahashi & Yamanaka, 2006; Takahashi et al., 2007). Now, iPSCs can routinely be generated from samples that are readily available from patients, e.g., skin biopsies or blood samples. The iPSCs can then further be differentiated toward the desired target cell populations, which can then be used as research material both in mechanistic studies and in treatment trials. Major advances have already been made, for example, in neurodegenerative disease and cardiac disease fields (Matsa, Burridge, & Wu, 2014; Peitz, Jungverdorben, & Brustle, 2013). We discuss here the use of iPSC-based disease modeling for patients with mtDNA mutations and mitochondrial disease.
2. INDUCED PLURIPOTENT STEM CELLS Embryonic stem (ES) cells have two unique properties: (1) they are able to self-renew and divide indefinitely, and (2) they are pluripotent and thus can differentiate to any cell type of an adult organism. iPSCs are somatic cells, e.g., adult fibroblasts, which have been driven backward in differentiation to an ES cell-like state (Takahashi & Yamanaka, 2006; Takahashi et al., 2007). iPSCs possess the two unique properties of ES cells,
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and thus they can be both propagated in culture indefinitely as iPSCs and differentiated basically to any adult cell type, such as neurons or cardiac cells, through in vitro differentiation. These two ES cell-like properties make iPSCs highly useful for different research and therapeutic purposes. Reprogramming of somatic cells to a pluripotent state was initially achieved by ectopic expression of four transcription factors highly expressed in ES cells, namely, Oct4, Sox2, Klf4, and c-Myc (Takahashi & Yamanaka, 2006; Takahashi et al., 2007). Since the original reports, different variations of the transgene cocktail, for example, omitting the c-Myc, which has oncogenic properties, have been reported to result in fully reprogrammed iPSCs (Nakagawa et al., 2008); however, the initial so-called Yamanaka-cocktail is still widely used because of its efficiency. In the original reports, the transgenes were introduced into the cells by retroviral infection (Takahashi & Yamanaka, 2006; Takahashi et al., 2007). With evolving technology, novel methods to introduce the reprogramming transgenes to host cells have been reported, including nonintegrative methods like Sendai virus or episomal plasmid vectors (Ban et al., 2011; Okita et al., 2011). Further, reprogramming of human cells to pluripotency has also been achieved by DNA-free methods using direct delivery of either mRNAs or proteins (Kim et al., 2009; Warren et al., 2010; Yoshioka et al., 2013). In addition to fibroblasts, iPSCs have been now generated from a variety of different cell types including blood cells and epithelial cells from urine samples (Staerk et al., 2010; Zhou et al., 2012).
2.1. iPSC-derived disease models One of the research fields most enhanced by iPS technology is disease modeling. iPSCs have already been generated from a variety of patients with different diseases and the list keeps growing (Park et al., 2008; Peitz et al., 2013). While animal models have been crucial for mechanistic studies of human disease pathology, models that thoroughly reproduce human pathology are rare and, for example, drug trials done in animal models do not always work in human patients (Inoue & Yamanaka, 2011). Challenges exist also in iPSC-based disease modeling. While generation of iPSCs is fairly robust, differentiation of the iPSCs to the desired cell type is not trivial and achieving pure target cell populations is at least difficult if not impossible. Also, a genetic defect in a cell may not always lead to functional manifestations, and different stressors may be needed to challenge the
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cultures and to mimic the physiological disease niches. Further, many lateonset diseases may not easily manifest their key features in short-term cultures. However, different stressors or nutrient deprivation may be used to enhance the aging process in the in vitro culture system. A good control setting is critical for all model systems, and this becomes even more evident with iPSC-based modeling, as, in addition to the normal biological variation between individuals, the variation between different iPSC clones may be remarkable. The current standard in the field is to use nondisease control lines together with gene-edited isogenic control lines (Soldner et al., 2011). While the isogenic control lines reduce noise significantly, multiple clones will still need to be analyzed to avoid off-target effects and clonal variation between different lines. Despite the challenges, iPSC-based disease modeling has already proven to be an efficient tool to study disease mechanisms and remarkable advances have been achieved. Successful model lines, which recapitulate manifestations seen in patient tissue, have been generated for various diseases. For example, underlying mechanisms and potential targets for therapy have been identified for severe human neurodegenerative disorders like amyotrophic lateral sclerosis and Alzheimer’s disease (Donnelly et al., 2013; Kondo et al., 2013) and successful drug tests have been done in iPSC-derived cardiac cells from patients with long QT syndrome (Lahti et al., 2012; Terrenoire et al., 2013).
3. MITOCHONDRIAL DISEASE Mitochondria are best known for their function in transforming the energy of nutrients in the form of ATP, through cellular oxidative phosphorylation. In addition to ATP production, mitochondria take part in several other cellular metabolic functions, and their signals affect whole cell signaling, including redox-dependent nutrient sensor and ROS signaling, cellular proliferation, differentiation, and programmed cell death. Mitochondria are under a dual genetic control. They possess multiple copies of their own DNA (mtDNA), which encodes 13 subunits of the mitochondrial respiratory chain (RC) enzymes as well as the mitochondrial tRNAs and rRNAs required for the synthesis of these proteins. All other mitochondrial proteins are encoded by the nuclear genome. Despite the small size of the mitochondrial genome, mtDNA mutations underlie a vast number of human inherited disorders. In western populations, mtDNA mutations cause a disease in every 1:5000 individuals
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(Skladal, Halliday, & Thorburn, 2003), and every 1:200 newborns carries a potentially pathogenic mtDNA mutation (Elliott, Samuels, Eden, Relton, & Chinnery, 2008). Over 200 mtDNA point mutations have to date been reported to underlie human disease (Ylikallio & Suomalainen, 2012). Clinical features of these diseases vary largely from infantile multisystem disorders to adult-onset myopathies and neurodegeneration, and in fact, mitochondrial disease can occur in any organ system with any age of onset (Ylikallio & Suomalainen, 2012). The manifestations may vary even in patients carrying exactly the same mutations: for example, m.3243A>G that leads to diabetes and deafness in some patients may lead to a brain phenotype and strokes or cardiac disease in others (Chae et al., 2004; Goto, Nonaka, & Horai, 1990; van den Ouweland et al., 1992). A typical feature of a pathogenic mtDNA mutation is heteroplasmy; the patient cells carry both normal and mutant mtDNA. The disease manifests when the mutant mtDNA amount exceeds a threshold, which may vary between tissues and different individuals (Ylikallio & Suomalainen, 2012). While segregation of mutated mitochondria and tissue-specific threshold levels are important factors in determining the clinical outcome of mtDNA mutations, other, as yet unidentified, factors also play an important role. This is evident, as even homoplasmic mtDNA mutations may cause variable clinical phenotypes (Gropman et al., 2004). To identify these additional factors and to study mechanisms underlying these disorders, good model systems are needed.
3.1. iPSCs from patients with mtDNA mutations Since other model systems for mtDNA disorders are scarce, high hopes are set for the use of iPSCs in studying these diseases. Initial concerns of whether cells that have a defect in ATP production were able to reprogram properly have been shown to be unwarranted, and iPSCs have been generated from patients with different mtDNA mutation types, both point mutations (Folmes et al., 2013; Fujikura et al., 2012; Ha¨ma¨la¨inen et al., 2013) and large mtDNA deletions (Cherry et al., 2013; Table 19.1). Independent of the mutation type or frequency, all reported iPSCs with mtDNA mutations have shown normal ES cell-like morphology and high expression of pluripotency marker genes. All reported lines have also been able to differentiate toward all three germ layers (Cherry et al., 2013; Folmes et al., 2013; Fujikura et al., 2012; Ha¨ma¨la¨inen et al., 2013). However, while cells harboring point mutations in either tRNA or RC protein-coding genes have
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Table 19.1 Published iPSCs with mtDNA mutations Cell types differentiated Disease Mutation from iPSCs References
Diabetes (MIDD)
A3243G
–
Fujikura et al. (2012)
MIDD/MELAS A3243G
Neurons
Ha¨ma¨la¨inen et al. (2013)
MELAS
G13513A
–
Folmes et al. (2013)
Pearson syndrome
10949_13449del Hematopoietic cells
Cherry et al. (2013)
MIDD, maternally inherited diabetes and deafness; MELAS, mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes.
shown normal efficiency and kinetics of reprogramming, the patient cells with large mtDNA deletions showed low reprogramming efficiency and the process was significantly slower than in control cells (Cherry et al., 2013). In all the studies reported so far, mtDNA heteroplasmy has shown specific segregation patterns, both during reprogramming and the subsequent culture and differentiation of the iPSCs. During reprogramming, all mtDNA mutations segregated in a bimodal fashion leading to iPS clones with either no detectable mutant mtDNA, or those with over 50% of the total mtDNA pool in a cell being mutated (Cherry et al., 2013; Folmes et al., 2013; Fujikura et al., 2012; Ha¨ma¨la¨inen et al., 2013). During culture of the iPSCs, the tRNA mutation (A3243G in tRNA-LeuUUR) remained with stable mutant mtDNA amount, whereas the protein-coding RC mutation (G13513A in ND5) frequency decreased. The large mtDNA deletion (10949_13449del affecting five genes; ND4, ND5, tRNA-LeuCUN, tRNA-SerAGY, and tRNA-His) was stable in certain iPSC clones, but decreased in others until undetectable. On the other hand, after reprogramming, during differentiation of the iPSCs, all mtDNA mutant cell lines retained stable heteroplasmy level: no significant changes in the heteroplasmy levels were found between the starting iPSCs and the differentiated cells in any of the studies (Folmes et al., 2013; Fujikura et al., 2012; Ha¨ma¨la¨inen et al., 2013). These segregation patterns of the mutated mtDNA fit well with data published previously from animal studies. The bimodal segregation of the mutations during reprogramming suggests a mitochondrial bottleneck, similar to the one seen in germ cells and during epiblast specification (Cao et al., 2007; Lee et al., 2012). Furthermore, purifying
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selection of mutations affecting protein-coding genes has been reported in mouse germ cells (Stewart et al., 2008). This seems to take place also in stem cells and fits well with the fact that the ND5 mutation frequency reduced during iPSC culture, while the tRNA-LeuUUR stayed stable (Folmes et al., 2013; Ha¨ma¨la¨inen et al., 2013). Due to the bimodal segregation of the mutations during reprogramming, it is possible to derive clones from one individual with both high mtDNA mutation frequency and no detectable mtDNA mutations. This is a major advantage, since it enables simultaneous generation of both disease model lines and their isogenic control lines, i.e., same nuclear background, from the same patients without additional gene editing. All the lines with mtDNA mutations reported so far have shown normal differentiation potential, and all heteroplasmic mtDNA variants have been stable during differentiation, allowing generation of desired target cell populations with high and low mutations frequencies. In addition to proving that generation of iPSC lines and their differentiation to the desired target cell types with high mtDNA mutation levels is feasible for different mtDNA mutation types, the initial studies have also proven that iPSC-derived cells manifest disease phenotypes typical for patient tissues, and that the manifestations are cell type specific. Cherry et al. showed that fibroblasts derived from bone marrow of a Pearson patient manifested a combined RC defect. iPSCs derived from these fibroblasts grew slowly, and this may at least partially explain the slow appearance of colonies during reprogramming of these cells. Also, the Pearson iPSCs with large mtDNA deletion manifested an RC deficiency. The cells showed low mitochondrial membrane potential, low cellular respiration, and partial compensation of this with increased glycolysis. However, differentiation of hematopoietic cells was not significantly altered between cells with different levels of mutant mtDNA. Among the in vitro differentiated erythroblasts, a high level of sideroblasts (abnormal erythroblasts with pathologic iron granule depositions in mitochondria) were seen specifically in the cultures with high frequency of deletion (Cherry et al., 2013), which is consistent with sideroplastic anemia being a hallmark of Pearson syndrome (Pearson et al., 1979). We showed in our study that while the fibroblasts with G3243A mutation in mtDNA manifested a mild combined RC defect, no RC defect existed in the iPSCs. The iPSCs with this tRNA-LeuUUR mutation grew normally and also the reprogramming kinetics was similar to control cells (Ha¨ma¨la¨inen et al., 2013). Further, we showed that in vitro differentiated neurons with high mtDNA mutation frequency
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manifested an isolated Complex I deficiency, which is typical also for patient tissues (Ciafaloni et al., 1992; Goto et al., 1992). Our data clearly show that manifestation of the RC defect is dependent on the cellular context, and stresses the importance of studying the relevant cell types for the specific diseases.
3.2. Utilization of iPSCs as mtDNA disease models iPSCs provide a powerful tool for studying mitochondrial disease, since they can be differentiated toward basically any adult cell type. Apart from neuronal and hematopoietic cells which have currently been generated from iPSCs with mtDNA mutations (Cherry et al., 2013; Ha¨ma¨la¨inen et al., 2013), it is also possible to derive many other cell types that are relevant for mtDNA disease from these same cells. Directed differentiation of iPSCs toward, for example, cardiac cells (Lahti et al., 2012; Terrenoire et al., 2013), retinal pigment epithelia (Kamao et al., 2014; Krohne et al., 2012), hepatic cells (Si-Tayeb et al., 2010; Sullivan et al., 2010), and pancreatic β-cells (Shahjalal et al., 2014) has already been successfully achieved. Apart from generating desired target cell types through directed differentiation protocols, iPSCs can be differentiated through teratoma formation. This does not allow for controlling the outcome of the differentiation, but will generate highly differentiated cells and tissues. The teratomas can be used to assess RC function in several different tissue types (Castro et al., 2010; Ha¨ma¨la¨inen et al., 2013). The bimodal segregation of mutant and wild-type mtDNA upon reprogramming resembles mtDNA bottleneck upon epiblast specification (Ha¨ma¨la¨inen et al., 2013; Lee et al., 2012), and this allows for using iPSCs and reprogramming to study mitochondrial bottleneck and the mechanisms that control mtDNA segregation during early embryonic development. As cell culture conditions still differ from mature tissues, some mitochondrial disorders do not manifest disease phenotype even when differentiated into neurons or cardiomyocytes, but the manifestation would require differentiation within a mature tissue. Obtaining good-quality iPSCs is a timeconsuming, laborious, and expensive process, and—similar to mouse models—the knowledge of whether the model is actually useful will only be available after iPSC generation and subsequent differentiation have been performed. Our experience for mtDNA mutations, however, has been promising as relevant, potentially pathologic physiological pathways mimicking patient tissues have been produced in culture.
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4. CONCLUSION iPSC-based disease modeling has provided a powerful tool for generating human patient-derived postmitotic cells, and especially so for heteroplasmic mtDNA mutations, which otherwise lack proper disease models (Fig. 19.1). iPSC-derived materials have already been successfully used for both mechanistic studies and treatment trials. It is clear that iPSC line generation from cells with mtDNA mutations is feasible. Furthermore, the bimodal segregation of mutant mtDNA upon reprogramming results in simultaneous generation of iPSC clones with high mutation frequencies as well as isogenic control lines with no detectable mutations. The heteroplasmy remains stable after reprogramming, enabling derivation of desired target cells, such as neurons or cardiomyocytes, with high or low mutation levels. The initial studies have also shown that iPSC-derived cells, hematopoietic cells from a Pearson patient, as well as neurons from an MELAS
Figure 19.1 iPSC-based disease modeling for disorders with mtDNA mutations. Fibroblasts (or other cell types) from patients can be reprogrammed to an induced pluripotent state. During reprogramming, heteroplasmic mtDNA mutations segregate giving rise to cells with either high or low mutation frequency. These cells can then be used as both disease models and isogenic controls for the disease lines. The cells can further be differentiated to any desired target cell type where tissue-specific disease manifestations can be studied. This provides a powerful tool to study disease mechanisms and to test potential therapeutic agents.
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patient manifest symptoms typically seen in patient tissues. These data suggest that iPSCs will provide a powerful tool to studying disorders with heteroplasmic mtDNA mutations.
5. GENERATION OF iPSCs FROM mtDNA DISEASE PATIENTS Generation of iPSCs from fibroblasts can be achieved by several different methods. We have used both retroviral (Takahashi et al., 2007) and episomal (Okita et al., 2011) methods and describe here the episomal plasmid method originally published by Okita and Yamanaka (Okita et al., 2013).
5.1. Materials Patient fibroblasts from skin biopsy. Episomal plasmids from Addgene: 27077 (pCXLE-hOCT3/4-shp53-F), 27078 (pCXLE-hSK), and 27079 (pCXLE-hMLN). Mouse embryonic fibroblast (MEF) feeder cells. Prepared from 12.5dpc ICR embryos, mitotically arrested with mitomycin-c treatment. Fibroblast culture media: DMEM (high glucose), glutamax, penicillin/ streptomycin, supplemented with 10% FBS. hES media: KO-DMEM, nonessential amino acids, glutamax, betamercaptoethanol (0.1 mM), and penicillin/streptomycin, supplemented with 20% KOSR (Life Technologies, Grand Island, NY, USA) and 10 ng/ml bFGF: used during iPSC establishment Essential eight medium (Life technologies): used during iPSC culture. Uridine Phosphate-buffered saline (PBS), Mg2+, and Ca2+ free TrypLE Select (Life Technologies) EDTA 0.5 mM Gelatin 0.1% Matrigel (BD Biosciences, Bedford, MA, USA) Trypan blue Freezing media (E8 + 10% DMSO) Hematocytometer or automated cell counter Electroporator e.g., Neon Transfection system (Life Technologies, Carlsbad, CA, USA) 15-ml centrifuge tube 6-cm culture dishes
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6-well culture dishes 4-well culture dishes scalpels cryovials
5.2. Protocol 1. Expand low passage fibroblasts (see Notes 1 and 2) in an appropriately sized dish in fibroblast medium. One 6-cm dish can yield about 1–1.5 106 cells. Once the desired cell number is reached, proceed to electroporation. 2. Prepare one 6-cm plate per cell line. Apply gelatin (0.1%) to the surface of the dish, aspirate, and allow to dry uncovered in the biosafety cabinet. Add fibroblast media (see Note 3) and put the plates in 37 C; 5% CO2. 3. When the fibroblasts reach 70–80% confluency (see Note 4), harvest them using enzymatic dissociation with TrypLE (5 min at 37 C), collect by centrifugation at 750 rpm for 3 min, resuspend in fresh medium, and count viable cells using trypan blue exclusion and hematocytometer or automated cell counter. 4. Collect 700,000 cells, wash with PBS, and resuspend in 120 μl of buffer R. 5. Mix 1 μg of each of the three plasmids pCXLE-hOCT3/4-shp53-F (27077), pCXLE-hSK (27078), and pCXLE-hMLN (27079) (all from Addgene) with the cells. Electroporate according to the system instructions (see Note 5) with 1650 V, 10 mS, and three pulses. Transfer the cells directly from the tip to the freshly prepared 6-cm dishes. Culture overnight in 37 C; 5% CO2. This is considered as day 1. 6. On days 2 and 4 feed the cells with fresh fibroblast media. 7. On day 5 prepare feeder plates for passaging the fibroblasts. Apply gelatin (0.1%) to the surface of a 6-well dish, aspirate, and allow to dry uncovered in the biosafety cabinet. Plate 100,000 mitotically inactivated MEFs per well in fibroblast media and let the cells adhere overnight in 37 C; 5% CO2. 8. On day 6 split the cells to the feeder cells. Change fresh media to the feeder cell plates. Harvest electroporated fibroblasts using TrypLE (5 min at 37 C), collect by centrifugation at 750 rpm for 3 min, resuspend in fresh fibroblast medium, and count viable cells. Plate 50,000–100,000 cells per well (see Note 6) on the feeder cells.
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9. On day 7 change to hES media and feed every second day with fresh media. Monitor the wells for appearance of characteristic iPSC growth foci which typically become visible after 2 weeks in culture (Fig. 19.2). 10. Colonies suitable for picking may form as early as day 20; however, clones will typically continue to arise until days 28–35. 11. Pick colonies mechanically, dividing large colonies into small pieces with a scalpel, a pulled glass pipette, or 30-gauge needle. Plate the picked colonies on freshly prepared 4-well dishes (see Note 7) with feeder cells and hES media. 12. A few initial passages should be done mechanically and the cells kept on feeder cells with hES media (see Note 8). Typical colony types are presented in Fig. 19.2. 13. After establishing the lines, the cells can be transferred to Matrigelcoated plates and cultured in E8 media using EDTA for passaging. Coat the plates with Matrigel and equilibrate to RT (1–2 h). Prewarm E8 medium at RT. 14. Aspirate the medium and rinse the cells with PBS. Add 0.5 mM EDTA and incubate at RT for 5–8 min. Proceed when the cells start to separate and round up. Aspirate the EDTA solution and add E8 to the dish. Remove the cells from the well by gently squirting medium and pipetting the colonies up but do not scrape the cells from the dish. Collect cells in a 15-ml conical tube. Add E8 to a matrigel-coated plate and plate the cells in a desired ratio. 15. Grow the cells in 37 C, 5% CO2 incubator and feed daily or every second day. Passage when needed. 16. Once the iPSC lines have been established, they can be frozen down. Prepare freezing media by adding 10% DMSO to E8 media. Place on ice until use.
Figure 19.2 Typical colony types present in early passage iPSC lines. (A) A perfect colony with clearly defined edges and high cell density. (B) A moderate quality colony with lower cell density. (C) A differentiating colony. The dense part with defined borders can be cut out and cultured further. (D) Differentiated cells.
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17. Treat the cells with EDTA as when splitting, but instead of media add 1 ml of ice-cold freezing media to each well. Remove the cells by gently squirting the colonies from the well. Collect the cells in cryovials, resuspend gently, and place on ice. Transfer first to 80 C and after 24 h to 150 C or liquid nitrogen for longer storage.
5.3. Notes 1. To avoid selecting against mtDNA mutations, it is necessary to include uridine (200 μM) and pyruvate to the culture media during all stages of cell culture (King & Attardi, 1989). 2. A high growth rate of the starting fibroblasts is critical for good reprogramming efficiency. 3. Antibiotics are optional in all culture media; however, immediately after electroporation, it is best to culture the cells overnight without antibiotics, to allow them to recover from the electroporation without any additional stress. 4. The cells should be in an exponential growth phase. 5. For electroporation we use a Neon electroporation system (Invitrogen), but other systems should work as well. The method is explained for Neon transfection system with 100 μl tips, but also 10 μl tips can be used. 6. The plating density depends on the growth rate of the fibroblasts. If the cells grow very fast, lower densities may be useful to prevent overgrowth of the cells during subsequent culture. If the plates become too dense, this will hinder the proper formation of the iPSC colonies and the fibroblasts may detach from the plates. 7. Even though the surface size of a 24-well dish is the same as a 4-well dish, we find it easier to use 4-well dishes, as these have lower edges, which will make the mechanical cutting easier. 8. During initial passaging, some differentiation and cell death may occur. This is normal, and mechanical cutting will help to select for the good pluripotent cells.
ACKNOWLEDGMENT Anu Suomalainen-Wartiovaara is thanked for valuable comments to the manuscript.
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