Human pluripotent stem cells in regenerative medicine: where do we stand?

in Reproduction
Correspondence should be addressed to C Spits; Email: Claudia.Spits@vub.be

Human pluripotent stem cells have the capacity to self-renew indefinitely and the ability to differentiate into all cell types of a human body. These characteristics instill them with an enormous promise in regenerative medicine, where they could be used in cell, tissue and even organ-based replacement therapy. In this review, we discuss their potential clinical applications and the advantages and pitfalls for the different types of human pluripotent stem cells to transition from the bench to the bedside. We provide an overview of the current clinical trials, and the specific challenges we are still facing, including immune compatibility, suboptimal differentiation, risk of tumor formation and genome instability.

Abstract

Human pluripotent stem cells have the capacity to self-renew indefinitely and the ability to differentiate into all cell types of a human body. These characteristics instill them with an enormous promise in regenerative medicine, where they could be used in cell, tissue and even organ-based replacement therapy. In this review, we discuss their potential clinical applications and the advantages and pitfalls for the different types of human pluripotent stem cells to transition from the bench to the bedside. We provide an overview of the current clinical trials, and the specific challenges we are still facing, including immune compatibility, suboptimal differentiation, risk of tumor formation and genome instability.

Human pluripotent stem cells (hPSC) have an unlimited capacity to self-renew and can differentiate into cells of all three embryonic germ layers, making them very appealing to the fields of regenerative medicine and in vitro disease modeling. There are two types of hPSC: human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC). The first are derived from early human embryos, while the second are obtained by reprogramming of somatic cells. By now, the derivation and culture of hPSC have found its way into many laboratories worldwide (Löser et al. 2010, Fraga et al. 2011), and these cells have become the basis of a very active research domain. The European Human Embryonic Stem Cell Registry contains 738 hESC and 1.822 hiPSC lines from 251 providers of 29 countries (http://www.hescreg.eu; Monthly Statistic Report To 01.08.2018), and, for instance, a PubMed search using the term ‘human pluripotent stem cells’ on 8/08/2018, yielded 27.179 results.

The first steps of hPSC in regenerative medicine

The greatest therapeutic promise of hPSC is to cure individuals by replacement of somatic cells that were lost as a result of degenerative disorders or injury (Fig. 1). To date, hPSCs have been successfully differentiated into virtually all cell types, with the exception of functional gametes, and are being considered for the treatment of numerous conditions, such as neurodegenerative diseases, macular degeneration, cardiac failure and type I diabetes mellitus (Trounson & DeWitt 2016). Tissues and organs with a more complex organization are more difficult to obtain, although different groups have developed hPSC-derived organoid cultures that recapitulate the inner ear, retina, brain, lung, kidney, pituitary gland, liver, small intestine and stomach (reviewed in Kretzschmar & Clevers 2016). Of these, some of the most remarkable examples are the organoids with human brain structures (Lancaster et al. 2013, Renner et al. 2017), the renal tissues with a kidney-like organization (Takasato et al. 2014, Morizane et al. 2015) and the retinal organoids (DiStefano et al. 2018, Li et al. 2018, Mclelland et al. 2018). Nevertheless, although these developments are very exciting, the clinical translation of hPSC is just kicking off. Table 1 provides an overview of the ongoing and completed clinical trials using hPSC-derived cell types.

Figure 1

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Figure 1

The potential applications of human pluripotent stem cells and the challenges in bringing them from the bench to the bedside. The figure illustrates the different steps in the process, from the derivation of hPSC lines to the transplantation of their differentiated progeny. HPSC can be obtained from donated human blastocysts (human embryonic stem cells, hESC), from blastocysts obtained by somatic cell nuclear transfer (SCNT-ESC) and by somatic cell reprogramming (induced pluripotent stem cells, hiPSC). These last two types are by definition immune compatible with the donor of the somatic cells. The cells are then kept in culture to obtain sufficient cells to initiate differentiation. The terminally differentiated cells may be used in regenerative medicine. The main current challenges in this process are indicated in red boxes. These include the choice among the different hPSC and immune compatibility, the susceptibility of hPSC to genome instability during prolonged culture, suboptimal differentiation and the complications arising during regenerative medicine.

Citation: Reproduction 156, 5; 10.1530/REP-18-0291

Table 1

On-going and completed clinical trials using hPSC-derived cells.

Trial numberStartLocationStatusNumber of patientsSummary of the study
Nbib12170082010Asterias Biotherapeutics, Inc., USACompleted5Phase I, safety study of GRNOPC1 in patients with neurologically complete, sub-acute, spinal cord injury
Nbib14698322011Astellas Pharma Inc, UKCompleted12Phase I/II, safety study of sub-retinal transplantation of hESC-RPE in SMD patients
Nbib13449932011Astellas Pharma Inc, USACompleted13Phase I/II, safety study of sub-retinal transplantation of hESC-RPE in patients with advanced Dry AMD
Nbib13450062011Astellas Pharma Inc, USACompleted13Phase I/II, safety study of sub-retinal transplantation of hESC-RPE in SMD patients
Nbib16748292012CHABiotech CO., Ltd, KoreaRecruitingEstimated enrolment: 12Phase I/IIa, safety study of sub-retinal transplantation of hESC-RPE in patients with advanced Dry AMD
Nbib16255592012CHABiotech CO., Ltd, KoreaUnknownEstimated enrolment: 3Phase I, safety study of sub-retinal transplantation of hESC-RPE in SMD patients
Nbib16912612012Pfizer, UKSuspendedEstimated enrolment: 10Phase I, safety and feasibility study of implantation of hESC-RPE in patients with acute wet AMD and recent rapid vision decline
Nbib24633442012Astellas Institute for Regenerative Medicine, USAActive, not recruiting11Long-term follow up to a phase I/II, safety study of sub-retinal transplantation of hESC-RPE in advanced dry AMD
Nbib20579002013Assistance Publique – Hôpitaux de Paris, FranceCompleted10Feasibility and safety study of transplantation of hESC-derived CD15+ Isl-1+ progenitors in severe heart failure
Nbib29419912013Astellas Institute for Regenerative Medicine, UKActive, not recruiting11Follow-up to 5 years of a phase I/II, safety study of sub-retinal transplantation of hESC-RPE in SMD patients
Nbib21221592014University of California, Los Angeles, USAWithdrawnN/APhase I/II, safety study of sub-retinal transplantation of hESC-RPE in patients with geographic atrophy secondary to MMD
Nbib22393542014ViaCyte, USAActive, not recruitingEstimated enrolment: 65Phase I/II study with two cohorts to evaluate the safety, tolerability, and efficacy of various doses of VC-01 combination product in subjects with type 1 diabetes mellitus
UMIN0000119292014Highway program for realization of regenerative medicine and others, JapanOn hold2Phase 1 Clinical Trial for the treatment of wet AMD Using an autologous hiPSC-derived RPE
Nbib22860892015Cell Cure Neurosciences Ltd., USA and IsraelRecruitingEstimated enrolment: 24Phase I/IIa dose escalation safety and efficacy study of hESC-RPE transplanted subretinally in patients with advanced dry AMD
Nbib23021572015Asterias Biotherapeutics, Inc., USAActive, not recruitingEstimated enrolment: 35A Phase 1/2a dose escalation study of AST-OPC1 in subjects with cervical sensorimotor complete spinal cord injury
Nbib24456122015Astellas Pharma Inc, USAActive, not recruiting13Long-term follow up of sub-retinal transplantation of hESC-RPE in SMD patients
Nbib24633442015Astellas Pharma Inc, USAActive, not recruiting11Long-term follow up to a phase I/II, open-label, multi-center, prospective study to determine the safety and tolerability of sub-retinal transplantation of hESC-RPE in patients with advanced dry AMD
Nbib24649562015Moorfields Eye Hospital NHS Foundation Trust, UKUnknownEstimated enrolment: 10Feasibility of production of iPSC-derived RPE cells fulfilling regulatory requirements for transplantation in dry AMD
Nbib27497342015Southwest Hospital, ChinaActive, not recruitingEstimated enrolment: 15Clinical study of sub-retinal transplantation of hESC-RPE in AMD and SMD
Nbib29035762015Federal University of São Paulo, BrazilRecruitingEstimated enrolment: 18Phase I/II clinical trial using stem cell-derived RPE implantation in patients with AMD, SMD and exudative AMD
Nbib16912612015Pfizer, University College London, UKActive, not recruiting2Phase I, safety and feasibility study of implantation of hESC-RPE in subjects with acute wet AMD and recent rapid vision decline
Nbib25906922015Regenerative Patch Technologies, LLC, USAActive, not recruiting11Phase I/IIa safety study of sub-retinal implantation of hESC-RPE on a polymeric substrate in subjects with advanced, dry AMD
Nbib33050292016CHA University, KoreaEnrolling by invitationEstimated enrolment: 3Safety study of sub-retinal transplantation of SCNT-hES-RPE cells in patients with advanced dry AMD
Nbib31021382016Pfizer, UKActive, not recruiting2Long-term safety follow up study following transplantation of hESC-RPE in subjects with acute wet AMD and recent rapid vision decline
Nbib30464072017Chinese Academy of SciencesThe First Affiliated Hospital of Zhengzhou University, ChinaRecruitingEstimated enrolment: 10A Phase I/II safety study in the treatment of Dry AMD with hESC-RPE
Nbib31196362017Chinese Academy of SciencesThe First Affiliated Hospital of Zhengzhou University, ChinaRecruitingEstimated enrolment: 50A Phase I/II, safety and efficacy study of striatum transplantation of hESC-derived neural precursor cells in patients with Parkinson’s disease
Nbib27554282018Chinese Academy of SciencesBeijing Tongren Hospital, ChinaRecruitingEstimated enrolment: 10Safety and Efficacy of Sub-retinal Transplantation of hESC-RPE in patients with dry AMD

AMD, age-related macular degeneration; hESC-RPE, human embryonic stem cell derived retinal pigmented epithelial cells; MMD, myopic macular degeneration; SCNT-hES-RPE, somatic cell nuclear transfer human embryonic stem cell-derived retinal pigmented epithelial cells; SMD, Stargardt’s macular dystrophy; UK, United Kingdom; USA, United States of America.

Neural cell types were among the first to be obtained through directed differentiation of hESC. The first clinical trial of hPSC was a safety trial using hESC-derived oligodendrocyte progenitor cells to treat spinal cord injury. It was initiated in October 2010 by the Geron Corporation and halted 1 year later due to a change in business strategy, after transplantation of five patients who were either paraplegic or quadriplegic. This clinical trial was later taken over by Asterias Biotherapeutics Inc. and completed. The therapy caused no adverse events, but also no motor or sensory neurological improvements were observed (Ilic et al. 2015). More recently, the company initiated a new phase 1/2a dose escalation study using the oligodendrocyte progenitor cells in patients with cervical spinal cord injury. Last year, the company reported that 9 months after the treatment, three out of six of their patients showed motor level recovery of at least two levels. Patients without the treatment show comparable spontaneous recovery in 26% of cases (http://asteriasbiotherapeutics.com/).

The vast majority of the currently ongoing hPSC-based clinical trials involve the use of retinal pigmented epithelial (RPE) cells derived from hESC, hiPSC and somatic cell nuclear transfer (SCNT)-ESC to restore or improve vision in patients suffering from retinal degenerative diseases including acute age-related macular degeneration (AMD) and Stargardt’s macular dystrophy (SMD) (Table 1; Schwartz et al. 2012, 2015, Diniz et al. 2013, Song et al. 2015, Mandai et al. 2017). These diseases are characterized by a loss of RPE cells, which leads to the death of the photoreceptors causing loss of vision. The human eye has become the favored testing ground for hPSC-based therapies for several reasons. Particularly, the eye is an immunoprivileged site, it is easy to monitor and the current RPE differentiation protocols are very robust and yield a pure cell population.

In 2013, the Riken Center for Developmental Biology initiated the first clinical research involving the use of hiPSC-derived cells in humans. A first patient suffering from AMD was transplanted with RPEs generated using hiPSC derived from her own skin cells. Unfortunately, the study was suspended in 2015 due to the identification of potentially harmful mutations in the second candidate’s hiPSC and the RPE cells derived from them (Mandai et al. 2017). The first successfully completed phase I clinical trial with hESC-derived cells concluded on September 2014 in England with the transplantation of hESC-derived RPE sheets into 18 patients, nine affected with AMD and nine with SMD. The clinical trial did not only show the feasibility and safety of the procedure but also revealed an increase in the sub-retinal pigmentation in 13 out of 18 treated patients (Schwartz et al. 2015), and an improvement in visual acuity of at least 15 letters in eight of 18 patients during the first year after surgery, with no adverse effects seen in any of them (Schwartz et al. 2015).

A third ectodermal cell type that is making its way into the clinical trials are stem cell-derived dopaminergic neurons and neural precursor cells for the treatment of Parkinson’s disease. Parkinson’s disease is a neurodegenerative disorder caused by the death of ventral mesencephalic dopaminergic neurons. Transplantation of fetal dopaminergic neurons in Parkinson’s disease patients, although yielding variable success, has demonstrated the feasibility of cell-based therapy in this disease. Over the years, several groups have proven that hPSC are a potentially sound source of this specific cell type, and several pre-clinical studies have been carried out in rodent and primate models (reviewed in Lindvall 2016). Very recently, the Chinese Academy of Sciences, in collaboration with the first affiliated hospital of Zhengzhou university, have started a phase I/II, open-label study to assess the safety and efficacy of striatum transplantation of hESC-derived neural precursor cells in patients with Parkinson’s disease. Specifically, the study will enroll 50 patients for cell injection, administering a single dose of neural precursor cells by stereotaxic intra-striatal injection (Wang et al. 2018).

Human ESC-derived cardiomyocytes have been successfully obtained either by directed differentiation (Laflamme et al. 2007) or via embryoid bodies formation (Caspi et al. 2007). They are also being investigated for the clinical application to support heart regeneration, and pre-clinical studies in small-animal models such as mice and rats have shown favorable results (Caspi et al. 2007, Shiba et al. 2012). Using a non-human primate model of myocardial ischemia, Chong et al. reported that these cells appear to fully integrate and regenerate infarcted hearts (Chong et al. 2014). Menasché and coworkers started a phase I clinical trial for transplantation of purified CD15+ Isl-1+ hESC-derived cardiac progenitors to six patients. A median dose of 8.2 million of highly purified hESC-derived cardiovascular progenitors were embedded in a biocompatible fibrin patch and transplanted into the infarcted area of the heart of patients with severely impaired cardiac function when the patients undergo scheduled coronary artery bypass surgery or mitral valve procedures (Menasché et al. 2014). This clinical trial was recently completed and demonstrated the technical feasibility of producing clinical-grade hESC-derived cardiovascular progenitors and supports their short- and medium-term safety, thereby setting the grounds for adequately powered efficacy studies (Menasché et al. 2018).

Another application that has reached the clinical trials is the use of hESC-derived pancreatic beta cells or pancreatic precursors to treat individuals with type 1 diabetes. Different research groups have obtained pancreatic endoderm that can efficiently generate glucose-responsive, insulin-secreting endocrine cells after transplantation (Kroon et al. 2008, Sui et al. 2013, Kirk et al. 2014, Pagliuca et al. 2014). ViaCyte Inc., a biotech company working on the development of a stem cell therapy for treatment of type 1 diabetes, optimized a protocol for the scalable production of functional pancreatic progenitors from hESC (Schulz et al. 2012). Their approach is based on delivering pancreatic precursor cells subcutaneously in a device with a selectively porous cell-impermeable membrane. These precursor cells are designed to further differentiate and mature in vivo after surgical implantation, not only to fully functioning insulin-producing beta cells but also to other endocrine cell types that make up the normal human pancreatic islet (Kirk et al. 2014). An alternative approach is the generation of islet-like organoids from hESC that are functionally capable of glucose-responsive insulin secretion as well as therapeutic effects (Kim et al. 2016). Recently, Ameri and coworkers have identified GP2 as a specific marker of human pancreatic endoderm cells and demonstrated that GP2+ pancreatic endoderm cells efficiently differentiate into glucose-responsive insulin-producing cells. Isolation of the target cells using this cell surface marker has a further advantage that it can help to eliminate the residual undifferentiated hESC, thus offering a safer route toward the manufacture of endocrine cells for therapy (Ameri et al. 2017).

The challenging side of hPSC

Despite the first successes in closing the gap between the bench and bedside, there is still a number of significant hurdles to overcome for hPSC to reach their full and safe clinical potential. Figure 1 illustrates each challenge at the different steps from hPSC derivation to their transplantation to the patient. In the next sections, we discuss the most significant issues in detail.

Ethical issues

The main reservation that has been raised against the use of hESC is based on ethical grounds because the derivation of these cells requires the use of human embryos (Rosner et al. 2014). Conversely, it is possible to obtain hESC from single blastomeres of cleavage-stage embryos, this being a potential embryo-sparing approach (Klimanskaya et al. 2006, Geens et al. 2009). In this setting, one blastomere is biopsied from the embryo in the same manner as done for pre-implantation genetic diagnosis. While this blastomere could be then used to establish a hESC line, the rest of the embryo could further develop and be transferred to the patient. On the other hand, although technically possible, this approach is practically inviable, because it requires patients undergoing a fertility treatment to agree to a blastomere biopsy on their embryos, potentially compromising their chances of becoming pregnant.

Immune compatibility: hESC, SCNT-ESC and hiPSC

A second issue raised on the use of hESC for transplantation was the risk of immune rejection since they are allogeneic to the patients (Zhan et al. 2004, Simonson et al. 2015). Currently, there are two options to solve this problem. The first appeared with the development of cell reprogramming (Takahashi & Yamanaka 2006, Takahashi et al. 2007). Human iPSC are reprogrammed directly from patients’ somatic cells, and thus circumvent embryo destruction to establish hPSC lines and generate autologous hPSC. The second approach is the use of SCNT to generate patient-specific hESC lines (Tachibana et al. 2013, Chung et al. 2014). In this context, SCNT is performed by transferring the nucleus of a somatic cell to an enucleated human oocyte and the resulting embryos are then used for the derivation of ESC (SCNT-ESC) lines. It is important to keep in mind that SCNT is technically very challenging, and the derivation of ESC lines from human SCNT embryos has only recently been achieved (Tachibana et al. 2013, Chung et al. 2014).

Despite the strong similarities between hESC, hiPSC and SCNT-ESC, there is still debate on whether these cells are indeed equivalent both in biological terms and suitability for clinical applications (reviewed in Wolf et al. 2017). There are two main issues being discussed, namely (i) the differences in transcriptional and epigenetic landscape and (ii) the potential differences in mutational burden between these cells. For instance, Ma and coworkers compared genetically matched sets of hiPSC and SCNT-ESC to hESC and showed that the DNA methylation and transcriptional signatures of SCNT-ESC and hESC were remarkably similar and at the same time distinct to those of hiPSC, suggesting that SCNT-ESC undergoes more complete reprogramming than hiPSC (Ma et al. 2014). Using a similar approach, Johannesson et al. reported that gene expression and DNA methylation profiles of SCNT-ESC and hiPSC are similar (Johannesson et al. 2014). These contradictory results could be possibly partly explained by the use of different donor cell types, reprogramming methods and even time in culture, as the reprogramming process takes longer than first thought, and the cell lines settle in their pluripotent state with time (reviewed in Yoshihara et al. 2017a).

The cost of autologous hPSC

Despite the initial appeal of generating autologous hPSC for regenerative medicine, it is a non-neglectable issue that the generation of patient-specific hiPSC or SCNT-ESC would create a very expensive and lengthy treatment. This has made the use of hPSC banks for allogeneic treatment more appealing and realistic. In this line, scientists from Kyoto University have been establishing an hiPSC bank containing multiple clinical-grade hiPSC lines from donors homozygous for three human leukocyte antigens (HLA) loci: HLA-A, -B and -DR. This allows optimized matching with patients, reducing the degree of immune rejection of the allogeneic transplants (Masuda et al. 2014, Azuma & Yamanaka 2016). Different studies estimate that to offer an HLA-matched transplant to 90% of the Japanese population, a cell bank size of 50 (Nakatsuji et al. 2008) up to 140 (Okita et al. 2011) homozygous hiPSC lines would suffice. For a hESC bank, on the other hand, lines derived from 170 randomly selected donated embryos would provide at least one hESC line with a single mismatch at one locus or better match for 80% of patients (Nakajima et al. 2007).

Suboptimal differentiation

Another important issue is that the vast majority of differentiation protocols are not yet fully optimized. Most differentiation protocols result in a heterogeneous population of cells, frequently with only a small fraction of the desired cell type (D’Amour et al. 2006, Mfopou et al. 2010, Efthymiou et al. 2014). Often, these protocols try to mimic the microenvironment provided by the embryo. However, our understanding of these processes is still incomplete, and despite the use of matrices and specific growth factors to support the cells, the difference between the 3D environment of the embryo and the culture dish is still large. A strategy to partially counter this problem would be to derive and transplant progenitor cells rather than the fully differentiated cells (Kirk et al. 2014), assuming that the in vivo environment will assist the final differentiation steps. A further complication is added by the fact that many differentiation protocols show a wide variation in efficiency depending on which hPSC line is used as starting material. The leading hypothesis for these biases is genetic and epigenetic differences between the hPSC lines (extensively reviewed in Keller et al. 2018).

The epigenetic factor has received particular attention in hiPSC, with the idea of epigenetic memory. This concept stems from a number of works that investigated the differences in differentiation capacity of hiPSC and described them to epigenetic marks inherited from the cells of origin and that had not been correctly reset during the reprogramming process (Bar-Nur et al. 2011, Kim et al. 2011, Ohi et al. 2011). However, current knowledge suggests that the reprogramming process itself is prone to inducing epigenetic abnormalities (Tiemann et al. 2016) and that the main factor influencing the differentiation propensity is not an epigenetic memory as such, but the (epi)-genetic background of the donor of the source cells (reviewed in Keller et al. 2018).

The state of pluripotency of PSC lines is also emerging as an important inducer of differentiation bias. In mouse, ESC can exist in two states depending on the development stage of the embryos used for the derivation. Mouse naïve or ground state ESCs are derived from pre-implantation embryos, while mouse primed-state ESC are derived from post-implantation embryos. These two mouse ESC types have very different characteristics, including a lower differentiation bias in mouse-naïve ESC. In the human, hESC are derived only from pre-implantation embryos, but molecular analyses show that hESC are more similar to mouse primed-state ESC than to mouse-naïve ESC (Tesar et al. 2007, Nichols & Smith 2009, Hanna et al. 2010). Currently, the derivation of human-naïve ESC is possible through different methods, including the ectopic overexpression of the transcription factors and the use of tailored culture systems (reviewed in Weinberger et al. 2016). The most important advantage to human-naïve ESC is that they should display stable self-renewal capacity and survival, allowing efficient cell expansion and differentiation, and show less differentiation bias (Honda et al. 2013, Duggal et al. 2015). However, recent studies have confronted this notion by showing that naïve hPSC display a reduction in differentiation capacity and an increase in differentiation bias compared to the primed hPSC (Lee et al. 2017, Warrier et al. 2017). Furthermore, naïve hPSC would fail to generate mature cells, in contrast to their primed counterparts (Warrier et al. 2017).

An important problem caused by imperfect differentiation is the persistence of residual undifferentiated cells that may lead to tumor formation upon transplantation (Fujikawa et al. 2005, Blum & Benvenisty 2008, Sui et al. 2013). Even small numbers of remaining undifferentiated cells can lead to tumor formation (Lee et al. 2009) or the growth of immature tissues (Roy et al. 2006), making the achievement of a pure differentiated population of desired cell type prior to transplantation prerequisite. Several different attempts have been made to reduce the frequency of teratoma formation after transplantation, such as eliminating the residual undifferentiated cells by flow cytometry (Tang et al. 2011, Quintanilla et al. 2014), extending the in vitro differentiation culture period (Brederlau et al. 2006, Doi et al. 2012), reducing the number of transplanted cells (Lee et al. 2009) or treating the cell suspensions with agents that are specifically cytotoxic to undifferentiated cells prior to transplantation (Choo et al. 2008, Ben-David et al. 2013, Lee et al. 2013, Rosner et al. 2014, Mitsui et al. 2015). Nevertheless, and despite these efforts to eliminate these cells from the final differentiated product, the best solution would be to identify and tackle the causes of why these residual cells stay undifferentiated.

Genome instability

There is significant concern about the genetic and epigenetic integrity of hPSC. The increasing body of knowledge on this topic has shown that, up to now, it appears that any aspect of the genome of hPSC that is investigated, reveals forms de novo mutagenesis and instability.

It is by now well established that hPSCs that acquire chromosomal abnormalities that offer them with a selective advantage will outgrow and eventually take over the culture (reviewed in Lund et al. 2012, Nguyen et al. 2013). For example, more than 20% of the hPSC lines worldwide show a gain of a small region of 20q11.21 (Amps et al. 2011), which leads to overexpression of BCL2L1 (Bcl-xL), inhibiting the mitochondrial apoptosis pathway and conferring a strong selective advantage to the mutant cells (Avery et al. 2013, Nguyen et al. 2014). Remarkably, the 20q11.21 amplification is also common in cancer (Scotto et al. 2008, Beroukhim et al. 2010, Tabach et al. 2011), illustrating the link between culture adaptation and malignancy. For the other common chromosomal abnormalities appearing in hPSC after prolonged culture (such as gains of chromosomes 1, 12 and 17), the driver genes have not yet been conclusively established. The most compelling evidence is for the gain of chromosome 12, where the smallest common region of gain comprises the pluripotency regulator NANOG (Draper et al. 2004, Mayshar et al. 2010, Ben-David et al. 2014). HPSCs carrying this gain show decreased differentiation capacity and their gene expression profiles are similar to that of germ cell tumors and cells transgenically overexpressing NANOG (Ben-David et al. 2014).

Although the subject of much research, what causes and modulates the spontaneous mutagenesis of hPSC in culture has not yet been fully understood. It is known that hPSC undergo significant DNA damage in culture, resulting in different chromosomal gains and losses in individual cells, leading to mosaic cell populations (Jacobs et al. 2014), and it has been repeatedly suggested that hPSC culture conditions may play a significant role in this process. The two most investigated factors have been the method of passaging and the oxygen tension in the incubators. A number of reports have shown that aneuploidy more commonly appears when using enzymatic or chemical passaging methods (Buzzard et al. 2004, Draper et al. 2004, Ravi et al. 2005, Imreh et al. 2006), and that room oxygen tension results in an increase in spontaneous chromosome breaks and gross structural rearrangements (Forsyth et al. 2006, Lim et al. 2011). Recently, culture medium has been identified as a major influence on DNA damage and genomic instability (Jacobs et al. 2016, Bangalore et al. 2017). High-density culture in KnockOut Serum Replacement (KnockOut SR) containing media and on mouse feeders results in medium acidification, leading to increased DNA damage and chromosome abnormalities (Jacobs et al. 2016). Conversely, cells grown in mTeSR and Essential 8 media show higher levels of genotoxic stress and DNA sequence changes than their counterparts grown in KnockOut SR medium (Bangalore et al. 2017). Additionally, aneuploidy as such appears to also make the cells more prone to further genomic instability, due to DNA replication stress, resulting in defective chromosome condensation and segregation (Lamm et al. 2016).

Next to acquired chromosomal changes, hiPSC have two additional sources of loss of genetic integrity: the reprogramming process and the genetic variation in the source cells, originating from somatic mutagenesis (reviewed in Yoshihara et al. 2017a). The first reports on the appearance of de novo CNVs, mostly deletions, in early passage hiPSC suggested that these mutations appeared during reprogramming process and that they progressively disappeared from the culture due to their deleterious effect on the cells (Hussein et al. 2011, Laurent et al. 2011). Later work showed, though, that at least half of these already existed as low-frequency variants in the source cells (Abyzov et al. 2012). Point mutations have also been repeatedly reported in hiPSC (Gore et al. 2011, Ji et al. 2012, Young et al. 2012, Sugiura et al. 2014, Araki et al. 2017, Yoshihara et al. 2017b), and there is evidence that their genome has significantly higher point mutation rates as compared to that of hESC (Sugiura et al. 2014). Here again, the point mutations are suggested to have either pre-existed in the parental somatic cells or to be reprogramming associated (Gore et al. 2011, Ji et al. 2012, Young et al. 2012) and particularly occurring during the initiation step of cell lineage conversion (Sugiura et al. 2014, Araki et al. 2017, Yoshihara et al. 2017b). By all accounts, this significant level of point mutations is concerning, particularly when bearing in mind that both hESC and hiPSC, in the undifferentiated and differentiated state, have been recurrently found to carry TP53 missense mutations (Merkle et al. 2017). The consequences of this in the setting of regenerative medicine are potentially very damaging, as illustrated by the halted RIKEN clinical trial, upon identifying undisclosed genetic variants in their hPSC-derived cells (Garber 2015).

Finally, a last aspect that highlights how the somatic origin of hiPSC hampers their genetic integrity, are the findings regarding their mitochondrial genome. HESC show few variants in their mitochondrial DNA (mtDNA), at relatively low heteroplasmic loads, and are either inherited, or acquired during prolonged in vitro culture, but rarely are of high pathogenic potential (Maitra et al. 2005, Zambelli et al. 2018). In contrast, hiPSCs can display very high heteroplasmic loads of pathogenic mutations. By now, several groups have shown that these variants are found in the source cells (Mah et al. 2011, Kang et al. 2016, Perales-Clemente et al. 2016, Zambelli et al. 2018), that they originate from somatic mutagenesis, and correlate to the age of the cell donor (Kang et al. 2016). Although it is unclear what the exact impact of these mutations is on the functionality of the cells, studies from the field of disease modeling have provided evidence that they may induce defects in differentiation capacity and functionality of hPSC and hPSC-derived cells (Cherry et al. 2013, Folmes et al. 2013, Hatakeyama et al. 2015).

Taken together, there is substantial evidence indicating a susceptibility of hPSC to acquire mutations analogous to those found in cancers. Culture conditions during derivation, expansion and differentiation should be further optimized in such way that genetic instability is minimized, and hPSC and their derivatives should be subjected to genome-wide analyses, especially before clinical application, to avoid transplantation of what could be pre-cancerous cells. It is also clear that there is a lack of systematic studies addressing the risk of these mutations, and there are no guidelines to which clinicians and researchers can adhere (Andrews et al. 2017). At the moment, the COSMIC (Catalogue of Somatic Mutations In Cancer) database and the Shibata list from the Pharmaceuticals and Medical Devices Agency in Japan (http://www.pmda.go.jp/files/000152599.pdf#page=8) are the two suggested resources to predict the functionality of these variants and assess their potential impact on the safety of these cells.

Conclusions

Although holding the great potential in regenerative medicine, there is still a big gap to be filled for hPSC to reach their full potential. This review has provided an overview on the potential clinical applications, and the advantages and drawbacks of the different types of hPSC to transition from the bench to the bedside. In addition, we have also discussed the specific challenges that still need to be overcome, including immune compatibility, suboptimal differentiation, risk of tumor formation and genome instability in order to bring hPSC closer to the clinic.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.

Funding

This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

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    The potential applications of human pluripotent stem cells and the challenges in bringing them from the bench to the bedside. The figure illustrates the different steps in the process, from the derivation of hPSC lines to the transplantation of their differentiated progeny. HPSC can be obtained from donated human blastocysts (human embryonic stem cells, hESC), from blastocysts obtained by somatic cell nuclear transfer (SCNT-ESC) and by somatic cell reprogramming (induced pluripotent stem cells, hiPSC). These last two types are by definition immune compatible with the donor of the somatic cells. The cells are then kept in culture to obtain sufficient cells to initiate differentiation. The terminally differentiated cells may be used in regenerative medicine. The main current challenges in this process are indicated in red boxes. These include the choice among the different hPSC and immune compatibility, the susceptibility of hPSC to genome instability during prolonged culture, suboptimal differentiation and the complications arising during regenerative medicine.

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