AIMS Cell and Tissue Engineering, 1 (1): 31-46
DOI: 10.3934/celltissue.2017.1.31
Received: 07 April 2017
Accepted: 16 June 2017
Published: 19 June 2017
http://www.aimspress.com/journal/CTE

Review

Human Mutations Affecting Reprogramming into Induced Pluripotent
Stem Cells
Yohei Hayashi *Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan* Correspondence: Email: yohei.hayashi@md.tsukuba.ac.jp; Tel: 029-853-3929.
Abstract: The development of induced pluripotent stem cells (iPSCs) provides unprecedented
opportunities for life sciences, drug discovery, and regenerative medicine. iPSCs have been generated
from somatic cells in many patients with various genetic diseases carrying specific mutations. However,
the efficiency of iPSC generation is quite low. Less than 1% of human primary somatic cells can usuallyturn into iPSCs. Previous studies have revealed that cellular signaling pathways, epigenetic status, andcellular senescence were major barriers to iPSC generation. Serendipitously in some cases, humanmutations themselves affect the reprogramming efficiency of iPSC generation as well as cellular
phenotypes recapitulating their disease symptoms. Mutations, which cause altered DNA repair (e.g.,
Ataxia-Telangiectasia, fanconi anemia and DNA Ligase IV (LIG4) syndrome), premature aging (e.g.,
Hutchinson–Gilford progeria syndrome and Néstor–Guillermo progeria syndrome), altered telomere
homeostasis (e.g., dyskeratosis congenita), mitochondrial respiratory dysfunction, chromosomal
abnormalities, and fibrodysplasia ossificans progressiva, have all been shown to affect the
reprogramming efficiency of somatic cells to iPSCs. In this review, the effects of such mutations are
summarized and the methods which have been employed to rescue efficient iPSC generation frommutant cells is discussed. Although the mutations affecting reprogramming processes are rare, thesemutations have been invaluable to the elucidation of reprogramming mechanisms and to thedevelopment of improved reprogramming technologies.

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Keywords: Induced pluripotent stem cells; mutations; reprogramming; ataxia-telangiectasia;
fanconi anemia; dyskeratosis congenital; LIG4 syndrome; mitochondrial respiratory dysfunction;
fibrodysplasia ossificans progressive; p53; chromosomal abnormality

1. Introduction
Methods, by which to generate induced pluripotent stem cells (iPSCs), were developed bytransducing defined transcription factors, such as OCT4, SOX2, KLF4, and MYC, into somatic cells [1,2].
Such transcription factors regulate the expression of genes important for self-renewal and pluripotency.
However, only a small proportion of the transduced cells are reprogrammed to generate iPSCs
successfully [3], which is a major road block to applying this technology for biomedicine. Previous
studies have revealed several causes of the low efficiency of iPSC generation. iPSC generation requires
specific activation and/or inhibition of cell signaling pathways induced by various cytokines and/or
small chemicals [4,5]. The culture conditions during iPSC generation should be optimized.
Reprogramming to pluripotency involves the global changes of epigenetic modifications (e.g., gene
expression patterns, DNA methylation status, and histone modifications) specific to each somatic celltype; however, the changes can be incomplete in some iPSCs, which maintain the “epigenetic memory”
of original cell types [6–8]. P53- or p16-mediated cell senescence has been shown as a major barrier
against successful reprogramming into iPSCs [9–13]. Furthermore, although several other genes andmolecules have been shown to regulate the reprogramming process, the precise effects of each during
reprogramming remain unclear.
Disease-specific iPSCs have been generated from patients with a variety of genetic diseases witheither Mendelian or complex inheritance patterns and have offered an unprecedented opportunity torecapitulate both normal and pathologic human tissue formation in vitro, thereby enabling disease
investigation and drug development [14]. Serendipitously in some cases, these mutations also affect the
reprogramming processes. Such cases have been invaluable in elucidating reprogramming mechanisms
and developing improved reprogramming technologies. Here, I classify those reported to date into
biological mechanisms and/or pathways.
2. DNA repair
Ataxia-Telangiectasia (A-T; OMIM #208900) is a rare neurodegenerative disease characterized byearly-onset progressive cerebellar ataxia, eyes and skin telangiectasia, immunodeficiency, chromosomal
instability, hypersensitivity to ionizing radiation, and increased cancer risk [15]. A-T is caused by adefect in the ATM (Ataxia–telangiectasia mutated) gene [16], which produces a serine–threonine kinase
activated when cells are exposed to DNA double-strand breaks (DSBs) [17]. ATM phosphorylates many
proteins regulating cell cycle checkpoint, apoptotic responses, and DNA repair, including p53, Chk2,
BRCA1, RPAp34, H2AX, SMC1, FANCD2, Rad17, Artemis, and Nbs1. This activity initiates
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cell-cycle arrest at G1/S, intra-S and G2/M checkpoints and promotes DNA repair after DSBs [18].
Several groups have independently generated A-T iPSCs and demonstrated that neuronal cells
differentiated therefrom were excellent models to examine A-T-associated neurodegeneration [19–23].
The reprogramming efficiency from A-T patients’ somatic cells was markedly decreased compared tofibroblasts from healthy individuals [19,21,22]. Also, fibroblasts from Atm-deficient mice showedremarkably low efficiency of iPSC generation compared to somatic cells from wild-type mice [13,24].
These results suggested that ATM-mediated DNA-damage responses were important for reprogramming
toward pluripotent state. In order to rescue the decreased iPSC generation efficiency of A-T patients’cells, Bhatt et al., expressed BCL-xL (isoform Bcl-X(L) of BCL2L1), which markedly increased the
reprogramming efficiency of normal blood cells [25–27], together with reprogramming factors during
iPSC generation. This method allowed the generation of A-T iPSCs with high efficiency under
xeno- and feeder-free culture conditions [28]. Regarding the genomic integrity of ATM-deficient iPSCs,
a study showed that they were characterized by additional copy number variations (CNVs), suggestingthat ATM-mediated DNA-damage response was important for maintaining genomic integrity in
iPSCs [29]; however, other studies have shown that A-T iPSCs are chromosomally stable with rates ofnucleotide substitution during long-term cultures comparable to control iPSCs [21].
Dysfunctions in non-homologous-end-joining (NHEJ) repair of DNA DSBs cause several humansyndromes. DNA Ligase IV (LIG4) plays an essential role in NHEJ pathway [30,31]. Mutations in
LIG4 result in LIG4 syndrome (OMIM #606593), which is characterized by growth defects,
microcephaly, reduced number of blood cells, increased predisposition to leukemia, and variable
degrees of immunodeficiency [32–34]. The generation of LIG4 syndrome-specific iPSCs has beenreported in two independent studies [35,36]. These studies demonstrated that the impairment of
NEHJ-mediated-DSB repair in LIG4 iPSCs resulted in the accumulation of DSBs and enhanced
apoptosis. These studies also consistently reported the decreased efficiency of iPSC generation from
LIG4 syndrome patients’ cells. One of the studies showed that the complementation of wild-type LIG4
partially rescued the reprogramming efficiency [36]. This study also used patients’ cells with mutations
in genes encoding DNA-protein kinase catalytic subunit (DNA-PKcs; OMIM #615966), and Artemis(OMIM #602450). They reported that DNA-PKcs mutant cells showed a modest decrease inreprogramming efficiency whilst Artemis mutant cells showed comparable reprograming efficiency towild-type cells. Similarly, the reprograming efficiency of cells from individuals with mutations in
Cernunos/XRCC4-LIKE FACTOR (XLF; OMIM #611291) was similar to that of healthy donors [37].
Taken together, these results indicated a critical importance of the LIG4-mediated NHEJ pathway forsomatic cell reprogramming and self-renewal of iPSCs. However, there appears to be a spectrum of
reprogramming defects across humans with germline mutations in NHEJ factors. Fanconi anemia (FA;OMIM #227650) is a rare recessive, autosomal or X-linked, chromosomal instability disorder, causingbone marrow failure, and increased predisposition to develop malignancies [38,39]. FA is caused bymutations in any of the 15 genes identified so far, which are involved in FA pathway. Most of FA
pathway proteins form a unique nuclear protein complex that ubiquitinates FANCD2 and FANCI,
leading to the formation of DNA repair reaction [40,41]. FA patients’ cells displayed chromosomal
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instability and hypersensitivity to the DNA crosslinking agents that have been used for the diagnosis of
FA [42]. Several groups have independently generated FA-specific iPSCs from FANCA, FANCC,FANCD2, and FANCG mutant cells and demonstrated that hematopoietic lineage differentiation from
FA-specific iPSCs represented a good model system to recapitulate FA-associated diseasephenotypes [43–49]. All of these studies showed compromised generation or decreased efficiency of
FA iPSCs and indicated that activation of FA pathway proteins was critical in the reprogramming
processes. In order to rescue the generation of iPSCs from FA patients’ cells, a study reported that theoverexpression of human papillomavirus 16 (HPV16) E6 protein or p53 repression in combination of
reprogramming factors overcame barriers to reprogramming [46]. However, the resulting FA iPSCs
remained sensitive to the deficiency of the FA pathway and failed to self-renew. These results
suggested that FA pathway genes are essential for DNA-repair mechanisms during reprogrammingand for self-renewal of established pluripotent stem cells.
In summary, the DNA repair genes which affect the efficiency of reprogramming are shown in
Figure 1. The role of these genes in reprogramming largely remains elusive but examining further the
involvement of DNA repair pathways in iPSC reprogramming mechanisms would be fruitful for a more
complete understanding of the process.

Figure 1. A figure illustrating the DNA repair genes which mutations affectreprogramming efficiency.
3. Senescence
P53-mediated cell senescence is a major barrier against successful reprogramming into iPSCs using
p53-null mouse cells [9–13]. Although iPSCs from patients’ cells carrying germline mutations in p53,
which cause Li-Fraumeni Syndrome (LFS; OMIM #151623), have been generated using standard
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Yamanaka factors, the study did not report on the efficiency of reprogramming [50]. Another study
showed that the absence of p53 activity also contributed to the enhanced efficiency of iPS-like cell
generation from a cancer cell line, HCT116 [51]. The generation of iPS-like cells from HCT116 cells
was successful only when seven reprogramming factors were used; OCT4, SOX2, KLF4, LIN28 (alsoknown as LIN28A), nontransforming L-MYC, SV40 large T antigen (SV40LT) and small hairpin (sh)RNA against p53 [52]. These studies suggested that the inhibition of p53 was critical for the generationof iPS-like cells from cancer cells as well as from normal cells. Another study also demonstrated thatboth hypoxia and p53 deficiency increased iPS-like cell generation from HCT116 cells [53].
Hutchinson–Gilford progeria syndrome (HGPS; OMIM #176670) and Néstor–Guillermo progeria
syndrome (NGPS; #614008) are rare premature ageing diseases. HGPS is caused by a single point
mutation in the lamin A (LMNA) gene, resulting in the generation of progerin, a truncated splicing
mutant of lamin A [54]. Progerin accumulation leads to various aging-associated nuclear defects,
including disorganization of nuclear lamin A and heterochromatin loss. NGPS is caused by ahomozygous mutation in BANF1 (barrier-to-autointegration factor 1), resulting in the impairment of
post-mitotic nuclear envelope assembly by the loss of direct binding to chromatin, lamin A, andemerin [55,56]. Several studies have reported the generation of iPSCs from HGPS patients’ cells andhave successfully demonstrated that vascular smooth muscle cells (SMCs), mesenchymal stem cells(MSCs) or fibroblasts differentiated from HGPS-iPSCs recapitulated the age-associated alterations seenin HGPS, such as premature senescence [57–60]. Two of the studies reported that no iPSC colonies wereobtained from HGPS fibroblast cultures at late passages [57,58]. One of the studies also reported that the
reprogramming efficiency of HGPS fibroblasts was 4-fold lower than that of parental control fibroblasts
at earlier passages [57]. Another study reported that the reprogramming efficiency of NGPS fibroblasts
was significantly lower than that of control fibroblasts and showed that shRNA knockdown of BANF1
reduced the efficiency still further whilst ectopic expression of wild-type BANF1 rescued molecularalterations and reprogramming inefficiency in NGPS fibroblasts [61]. These results indicated the causal
effect of BANF1 mutations on iPSC reprogramming efficiency and, in further experiments,
demonstrated that genetic and pharmacological inhibition of NF-κB significantly increased the
reprogramming efficiency of fibroblasts from NGPS patients, HGPS patients, as well as from normal
aged-matched donors.
4. Telomere homeostasis
Dyskeratosis congenita (DC; OMIM #305000) is characterized by the defective maintenance ofblood, pulmonary and epidermal tissues [62,63]. DC is caused by mutations in genes controlling
telomere homeostasis, such as TERT, TERC, DKC1, or TINF2. Several studies have reported that there
were some differences in the kinetics of telomere maintenance in iPSCs from DC patients. Agarwal et al.
reported that DKC1 mutant (delL37) iPSCs had elongated telomeres compared to fibroblast cells [64],but other studies have reported telomere shortening and low TERC expression in TERC, TERT, or DKC1
mutant iPSCs [65–68]. Among them, two studies consistently demonstrated that the reprogramming
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efficiency of DKC1 mutant cells was lower, with slower kinetics compared to normal cells [64,66]. Oneof the studies also showed that the hypoxic conditions (5% O2) enabled successful reprogramming from
DKC1 mutant fibroblasts [64]. These results suggested that telomere maintenance was a prerequisite forsuccessful reprogramming into iPSCs and was associated with oxidative stress during reprogramming.
5. Chromosomal abnormalities
A study has shown that patient cells carrying 2.4 Mb duplication in chromosome 1 give rise to alower efficiency of iPSC generation than do normal, healthy cells [69]. When we generated iPSCs frompatients’ cells carrying ring chromosome 13, the efficiency of iPSC generation was much higher thanthat from healthy control cells [70]. Taken together, these results indicate that chromosomal
abnormalities affect reprogramming and the effect is dependent upon on the type of abnormality.
Although it might be hard to detect which genes are involved in the reprogramming process from thepatients’ cells carrying chromosomal abnormalities, these cells could be useful resources for the closer
examination of reprogramming mechanisms.
6. Mitochondrial respiration
Mitochondrial respiratory dysfunction caused by pathogenic mutations in mitochondrial tRNA
genes emerges only when mutant mitochondrial DNA (mtDNA) proportions exceed intrinsic pathogenic
thresholds. Thus, reprogramming efficiency was significantly decreased from fibroblasts with >90%
mutant mtDNA (m.3243A>G), whereas fibroblasts with less mutant mtDNA showed normal
reprogramming efficiency [71]. Another report has shown that mitochondrial respiratory defects causedby complex I mutations exerted only a modest decrease in reprogramming efficiency [72]. A recent
study has reported a correlation between mitochondrial spare respiratory capacity in somatic cells andtheir reprogramming efficiency [73]. These results suggest that high variety in reprogramming efficiency
among patient cells with mitochondrial respiratory dysfunction might be reflected by their different
mitochondrial spare respiratory capacity. Mechanistically, we recently reported that TCL1 expression
induced by KLF4 activates AKT to enhance glycolysis and inhibits mitochondrial polynucleotide
phosphorylase (PnPase) to suppress oxidative phosphorylation during iPSC generation [74]. Together,
these data indicate that mitochondrial respiratory activity is a key to successful reprogramming. Indeed,a low dose of resveratrol, a potent phenolic antioxidant found in grapes and red wine that also has
anti-proliferative and anti-inflammatory activity, facilitated efficient cellular reprogramming frommitochondrial respiratory dysfunction patient-derived fibroblasts into iPSCs [75].
7. BMP signaling
Fibrodysplasia ossificans progressiva (FOP; OMIM #135100) is a rare congenital diseasecharacterized by the abnormal activation of endochondral bone formation in soft tissues. Most FOP
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patients carry a missense mutation (617G>A) in Activin A Type I receptor (ACVR1), leading to thehyperactivation of bone morphogenetic protein (BMP)-SMAD signaling pathway [76]. We, and othergroups, have generated iPSCs from FOP patients’ cells and successfully recapitulated FOP phenotypes
in vitro [77–81]. BMP signaling has complicated roles in the maintenance of pluripotency. BMP
promotes the self-renewal of mouse embryonic stem (ES) cells [82,83] whilst, in contrast, BMP inhibits
the self-renewal of human pluripotent stem cells [84–88]. In mouse iPSC generation, BMP signaling
facilitates their reprogramming [89]. Thus, BMP signaling has positive effects on both the induction andself-renewal of mouse pluripotent stem cells. The role of BMP signaling in human iPSC generation still
remains elusive. One study has shown that fully-reprogrammed iPSCs could only be generated when
BMP signaling inhibitors were employed [77], whilst we found an increased efficiency of iPSC
generation from FOP patents’ cells in different culture conditions and cell densities [90]. Furthermore,
the efficiency of iPSC generation from normal fibroblasts was enhanced by transducing mutant ACVR1(617G>A) or SMAD1 or by adding BMP4 protein at early periods of reprogramming. In contrast,
adding BMP4 at later periods decreased iPSC generation efficiency. ID genes, transcriptional targets of
BMP-SMAD signaling, are critical for iPSC generation. We subsequently found that the BMP-SMAD-ID
signaling axis suppressed p16/INK4A-mediated cell senescence, a major barrier to reprogramming.
However, the positive effect was easily masked by the overgrowth of non-reprogrammed cells when
initial cell densities were too high, or by alteration in the periods of BMP treatment. Furthermore, thedifferences in the culture medium constituents may modulate the outcomes partly because recent studies
have shown that Activin A, which normally activates only TGF-SMAD signaling, contributed theactivation of BMP-SMAD signaling in FOP cells [91,92]. Collectively, iPSC generation from FOP
patient cells are useful for clarifying the roles of cell signaling pathways in reprogramming processes.
8. Future Perspectives: rescuing methods of reprogramming in mutant cells which show
decreased iPSC generation efficiency
Here, I summarize the methods used to rescue reprogramming of mutant cells which show
decreased iPSC generation efficiency.
(1) Transient overexpression of the wild-type genes.
This method is simple, straightforward, and valuable to prove that the mutations themselves cause
the decrease the reprogramming efficiency. However, it might also affect the outcome of diseasemodeling using iPSC-derived differentiated cells if the overexpressed genes remain in the
reprogrammed iPSCs. For example in LIG4 syndrome iPSC generation, a study has demonstrated thatthe transient overexpression of wild-type LIG4 genes partially rescued the reprogramming efficiency [36].
(2) Addition of reprogramming enhancer genes.
Many genes have been reported to increase the reprogramming efficiency [93,94]. It may be helpful
to use these genes to improve efficiency from mutant cells. However, the effects of these genes might be
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context-dependent. For example in A-T cases, BCL-xL was used to increase the reprogramming
efficiency of patient-derived blood cells [28]. It might be also useful to overexpress target genes during
reprogramming, which can relieve the clinical symptoms caused by specific mutations.
(3) Treatment with specific chemicals during reprogramming.
Many chemicals have been reported to increase reprogramming efficiency [95–97]. It may be
helpful to use such chemicals to improve efficiency. However, the effects of these chemicals might becontext-dependent and even poorly reproducible. It might be also useful to treat with chemicals whichhave been shown to relieve the clinical symptoms caused by specific mutations during reprogramming.
For example, a low dose of resveratrol can increase the efficient cellular reprogramming of themitochondrial respiratory dysfunction patient-derived fibroblasts into iPSCs [75]. Furthermore, someconventional culture conditions during reprogramming might be incompatible for specific mutant cells.
In these cases, culture components, such as feeder cells, growth factors, hormones, extracellular matrices,
vitamins, or amino acids, could be changed to improve the reprograming efficiency [90,98–100].
(4) Hypoxic conditions.
Hypoxic conditions increase the reprogramming efficiency from normal human somatic cells [101].
This method can be generally used but the changes might be too subtle to increase effectively the
reprogramming efficiency from mutant cells. For example, successful reprogramming from DC
fibroblasts was achieved under low oxygen conditions (5% O2) [64].
9. Summary
In this review, I introduce human mutations affecting reprogramming efficiency into iPSCs(summarized in Table 1). However, it remains largely unclear why and how these mutations affect the
reprogramming process. I also introduce methods which have been used to rescue iPSC generation frommutant cells showing reduced or compromised reprogramming efficiency and I believe that thesemethods will also be useful in establishing disease-specific iPSCs from patients’ cells carrying mutations
involved in the reprogramming process itself.
Table 1. A summary of human gene mutations affecting reprogramming efficiency and kinetics.
Biological Type/ Disease Name/Type
Pathway
DNA repair
Ataxia-Telangiectasia (A-T; OMIM #208900)


LIG4 Syndrome(OMIM #606593)
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Reprogramming Efficiency
4 % (homo) and 15 % (hetero) compared to control
fibroblast (Ref. 19)~1 % compared to control fibroblast (Ref. 21)
somewhat reduced efficiency and slower kinetics (6–9
weeks) (Ref. 22)
0.7–17% compared to control fibroblast (Ref. 35)
Less than 1% compared to control fibroblast (Ref. 36)
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DNA-PKcs mutation(OMEM #615966)
Artemis mutation(OMIM #602450)
Cernunos/XLF mutation (OMIM #611291)
Fanconi Anemia(FA; OMIM #227650)


Senescence

Telomere
homeostasis

Chromosomal
Abnormalities
Mitochondrial
respiration

BMP signaling

Hutchinson–Gilford

progeria syndrome(HGPS; OMIM #176670)

Néstor–Guillermo
progeria syndrome(NGPS; #614008)
Dyskeratosis congenita (DC; OMIM #305000)


Slightly decrease or unchanged (Ref. 36)
Unchanged to control fibroblast (Ref. 36)
Unchanged to control fibroblast (Ref. 37)
No iPSC colonies from unmodified patients’ fibroblasts(Ref. 43, 44, )Lower efficiency (~10%) to control fibroblasts (+p53
shRNA) and slower kinetics (~40 days) (Ref. 45)
0 to 0.005% to input cells (Ref. 47)
No colonies with OSKM combination under hypoxic
conditions, but 0.0003 to 0.008% with OSKM+LIN28 andshp53 episomal plasmids (Ref. 49)
No iPSC colonies were obtained at late passages (Ref.
64,65)
At earlier passages, the efficiency of reprogramming of
HGPS fibroblasts was 4-fold lower than parental control
fibroblasts (Ref. 64).
Threefold to eightfold reduction in the reprogramming
efficiency compared to control fibroblasts (Ref. 68)
0 to 0.015% to input cells with slower kinetics (24–48 days)(Ref. 52)
No colonies in 21% O2 conditions, but 0.001 to 0.04% in 5%
O2 conditions (Ref. 54)
Lower Eficiency (Ref. 69)

2.4 Mb duplication in 
chromosome 1
Ring 13 chromosome  Higher Efficiency (Ref. 70)
Mitochondrial respiratory
 Reprogramming efficiency was significantly decreased in
dysfunction
patient’s fibroblasts with >90% mutant mtDNA(m.3243A>G) that caused severe mitochondrial respiratory
dysfunction, whereas fibroblasts with less mutant mtDNA
load had normal reprogramming efficiency (Ref. 71)
 Only a modest decrease in reprogramming efficiency inmutant cells with mitochondrial respiratory defects causedby complex I mutations (Ref. 72).
 Correlation between mitochondrial spare respiratory
capacity in somatic cells and their reprogramming efficiency(Ref. 73)
Fibrodysplasia ossificans Only many differentiated colonies from FOP patients’ cells
progressiva
with normal culture conditions; however they obtained fully(FOP; OMIM #135100)
reprogrammed iPSCs only when BMP signaling inhibitors
were treated (Ref. 77)
 In different culture conditions and cell density, an increased
efficiency of iPSC generation from FOP patents’ cells (Ref. 90)

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Acknowledgements
YH declares that there is no conflict of interest regarding the publication of this paper.
This work was supported in part by grants from the Ministry of Education, Culture, Sports, Scienceand Technology of Japan (JSPS KAKENHI). The funding bodies had no role in decision to publish, or
preparation of the manuscript.
Conflict of Interest
All authors declare no conflicts of interest in this paper.
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