Direct Conversion of Cell Fate and Induced Endothelial Cells

Abstract

Advances in nuclear reprogramming technology have enabled the dedifferentiation and transdifferentiation of mammalian cells. Forced induction of the key transcription factors constituting a transcriptional network can convert cells back to their pluripotent status or directly to another cell fate without inducing pluripotency. To date, direct conversion to several cell types, including cardiomyocytes, various types of neurons, and pancreatic β-cells, has been reported. We previously demonstrated direct lineage reprogramming of adult fibroblasts into induced endothelial cells (iECs) in mice and humans. In contrast to induced pluripotent stem cells, for which there is consensus on the criteria defining pluripotency, such criteria have not yet been established in the field of direct conversion. We thus suggest that careful assessment of the status of converted cells using genetic and epigenetic profiling, various functional assays, and the use of multiple readouts is essential to determine successful conversion. As direct conversion does not go through pluripotent status, this technique can be utilized for therapeutic purposes without the risk of tumorigenesis. Further, direct conversion can be induced in vivo by gene delivery to the target tissue or organ in situ. Thus, direct conversion technology can be developed into cell therapy or gene therapy for regenerative purposes. Here, we review the potential and future directions of direct cell fate conversion and iECs.

Historical Development of Nuclear Reprogramming

In 1957, Waddington proposed an epigenetic landscape model describing the process of cellular differentiation during normal embryonic development.¹ This model depicted a cell during terminal differentiation as a marble rolling down a hill into different grooves on the slope (Figure 1). It represents the classical dogma of cell fate determination that mammalian development is a unidirectional and irreversible process.

Figure 1.

Waddington’s epigenetic landscape. (A) The original model depicts cellular differentiation as a unidirectional, irreversible process. With accumulated evidence for nuclear reprogramming, the revised model now shows that cell fate can in fact be reset to the pluripotent status (B), and can also be directly converted into another fate (C).

This dogma has been challenged first by Gurdon et al in 1958² and later by Wilmut et al in 1996,³ who demonstrated cloning through somatic cell nuclear transfer (SCNT). In 1987, Lassar and colleagues presented another challenge to the dogma by their finding that ectopic expression of MYOD1 complementary deoxyribonucleic acid (DNA) directly converted fibroblasts to myoblasts.⁴ This pioneering work first demonstrated that cell fate conversion through transdifferentiation is feasible in mammalian cells. Since then, some studies have shown that direct conversion between closely related lineages, such as from glial cells to neurons and from B cells to macrophages, could be achieved by the ectopic induction of a single transcription factor.^5–8 In the early 2000s, somatic cells such as fibroblasts and thymocytes were found to be reprogrammed to an embryonic state after fusion with embryonic stem cells (ESCs).^9,10 This finding implies that certain unidentified factors can reprogram the nucleus of a somatic cell in an epigenetic manner.

Inspired by previous studies of nuclear reprogramming through SCNT,^2,3 forced induction of a single transcription factor,⁴ and ESC fusion,^9,10 Yamanaka and his colleague in their revolutionary work in 2006, demonstrated that overexpression of 4 transcription factors (OCT3/4, SOX2, KLF4, and c-MYC) selected from 24 ESC-specific genes dedifferentiated fibroblasts back to pluripotent stem cells.¹¹ These induced pluripotent stem cells (iPSCs) showed that only a handful of transcription factors can reconstitute the entire transcriptional network defining cellular identity, resulting in cell fate conversion.^11,12

The seminal work on dedifferentiation¹¹ attracted scientists back to the field of lineage reprogramming. In 2008, Melton and colleagues reported in vivo reprogramming of adult pancreatic exocrine cells to their closely related endocrine β-cells by the transduction of 3 transcription factors (Ngn3, Pdx1, and Mafa).¹³ Finally, in 2010, Wernig and colleagues demonstrated the direct conversion of fibroblasts to developmentally unrelated neurons by overexpressing a combination of 3 transcription factors (Ascl1, Brn2, Myt1l).¹⁴ In the same year, Ieda et al also showed direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) using a combination of 3 transcription factors (Gata4, Mef2c, Tbx5).¹⁵ Subsequently, several groups have reported different examples of transdifferentiation such as hepatocytes and various subtypes of neurons (Figure 2, Table 1, Supplementary Table).^16–42 These achievements clearly demonstrated that cell fate determination is multidirectional and reversible (Figure 1).

Figure 2.

Representative examples of direct conversion in human cells.

Table 1. Summary of Direct Conversion in Humans

Original cell	Induced cell	Conversion factors	Reference
Fibroblast	Glutamatergic neuron	ASCL1, BRN2, MYT1L, NEUROD1	Pang et al (2011)16
Fibroblast	Dopaminergic neuron	ASCL1, NURR1, LMX1A	Caiazzo et al (2011)17
Fibroblast	Dopaminergic neuron	ASCL1, BRN2, MYT1L, LMX1A, FOXA2	Pfisterer et al (2011)18
Fibroblast	Motor neuron	BRN2, ASCL1, MYT1L, LHX3, HB9, ISL1, NGN2	Son et al (2011)19
Fibroblast	Neural stem cell	SOX2	Ring et al (2012)20
Fibroblast	Cardiomyocyte	GATA4, MEF2C, TBX5, ESRRG, MESP1, MYOCD, ZFPM2	Fu et al (2013)21
Fibroblast	Cardiomyocyte	GATA4, HAND2, MYOCD, TBX5, miR-1, miR-133	Nam et al (2013)22
Fibroblast	Hepatocyte	FOXA3, HNF1A, HNF4A	Huang et al (2014)23
Fibroblast	Neural crest	SOX10	Kim et al (2014)24
Fibroblast	Hepatocyte-like cell	HNF1A plus any 2 of the factors, FOXA1, FOXA3, or HNF4A	Simeonov et al (2014)25
Fibroblast	Melanocyte	MITF, SOX10, PAX3	Yang et al (2014)26
Fibroblast	Retinal pigment epithelium	CRX, PAX6, MITF-A, OTX2, NRL, RAX	Zhang et al (2014)27
Fibroblast	Osteoblast	RUNX2, OSX, OCT4, L-MYC	Yamamoto et al (2015)28
Fibroblast	Neural stem cell	HMGA2, let-7b	Yu et al (2015)29
Fibroblast	Renal tubular epithelial cell	EMX2, HNF1B, HNF4A, PAX8	Kaminski et al (2016)30
Fibroblast	Megakaryocyte progenitor	GATA1, GATA2, RUNX1, LMO2, TAL1, C-MYC	Pulecio et al (2016)31
Fibroblast	Serotonergic neuron	ASCL1, FOXA2, LMX1B, FEV + p53 suppression	Xu et al (2016)32
Fibroblast	Excitatory cortical neuron	BRN2, MYT1L, FEZF2	Miskinyte et al (2017)33
Fibroblast	Hepatocyte-like cell	ATF5, PROX1, FOXA2, FOXA3, HNF4A	Nakamori et al (2017)34
Fibroblast	Motor neuron	ASCL1, ISL1, NEUROD1, BRN2, HB9, LHX3, MYT1L, NGN2	Zhang et al (2017)35
Fibroblast	Skeletal muscle progenitor	MyoD + TGFb inhibition, WNT & RTK activation, Col I	Boularaoui et al (2018)36
T cell	Neuron	BRN2, ASCL1, MYT1L, NGN2	Tanabe et al (2018)37
Fibroblast	Neural stem cell	PTF1A	Xiao et al (2018)38
Fibroblast	Hepatocyte	HNF4A, HNA1A, FOXA3	Ballester et al (2019)39
Fibroblast	Corneal epithelial cell	PAX6, OVOL2, KLF4	Kitazawa et al (2019)40
Fibroblast	Neuron	miR-9/9, 124 + KLF4, KLF5, 7SK	Cates et al (2021)41
Fibroblast	Endothelial cell	ER71, KLF2, TAL1 + siTWIST1 + rosiglitazone	Han et al (2021)42

Induced Endothelial Cells Directly Converted From Adult Fibroblasts

Building on the previous work, we also conceived the idea of transforming fibroblasts into functional endothelial cells (ECs) by transduction of key transcription factors specific to ECs. To this end, we first selected 11 candidate genes that are key players during endothelial development, including (1) genes that are involved in the development of hemangioblasts from the mesoderm (Gata2, Fli1, Tal1); (2) Ets family members that are central regulators of endothelial development (Elf1, Erg, Fli1, Ets1, Er71); (3) essential coworkers of endothelial transcription (Foxc2, Foxo1, Lmo2); and (4) a gene that regulates endothelial functions (Klf2). Lentiviral transduction of the 11 candidate genes altogether into adult skin fibroblasts (SFBs) from Tie2 promoter-driven green fluorescent (GFP) mice resulted in GFP+ cells, implying that the endothelial program was activated in these fibroblasts.⁴³ Serial stepwise screening revealed 5 key factors (Foxo1, Er71, Klf2, Tal1, and Lmo2) that were necessary and sufficient for reprogramming SFBs into Tie2-GFP+ cells. The conversion efficiency of these 5 factors was 4%. During reprogramming, pluripotent markers such as Oct4 and Nanog were not activated. The sorted Tie2-GFP+ cells showed endothelial morphology and possessed endothelial functions, including capillary formation on Matrigel and NO production. We thus termed them as induced ECs (iECs). iECs have genetic and epigenetic profiles similar to those of functional ECs such as the Mile Sven 1 mouse EC line and primary cultured mouse lung ECs. To our knowledge, this was the first study to report the direct conversion of mouse fibroblasts into functional ECs.⁴³ Each of the 5 key factors plays a crucial role in endothelial specification. Er71 is a key transcription factor orchestrating endothelial development,⁴⁴ and directly binds to the promoter region of EC-specific genes such as vascular endothelial (VE)-cadherin, platelet endothelial cell adhesion molecule (Pecam)1, and VE growth factor receptor (Vegfr). Foxo1 is essential for normal vascular development, and its disruption causes embryonic lethality.⁴⁵ Klf2 is induced by shear stress and regulates the expression of genes maintaining vascular tone.^46,47 Tal1 is essential for both hematopoietic and endothelial development.⁴⁸ Lmo2 functions as a transcriptional cofactor without directly binding DNA, and associates with Tal1 as part of a multifactorial complex.⁴⁹ Notably, Tal1 and Lmo2 synergistically specify hemangioblasts from the mesoderm, and differentiate them into the blood or endothelial lineage depending on the presence or absence of Gata1.⁵⁰

Subsequently, we tried to convert adult human fibroblasts (aHDFs) into functional ECs.⁴² This time we used VE-cadherin as a readout for successful conversion, as it is more specific to ECs than Tie2.⁵¹ Interestingly, only 3 transcription factors (Er71, Klf2, and Tal1) were necessary and sufficient, and no additional factors were needed for reprogramming aHDFs into VE-cadherin+ cells. However, unexpectedly, these VE-cadherin+ cells were distinct from ECs based on their genetic profiles as assessed by whole transcriptome sequencing, even though the cells showed endothelial features such as in vitro tube formation on Matrigel to some extent. We found that these cells did not fully express Pecam1, another stable marker of ECs. Thus, we further developed a protocol to derive VE-cadherin/Pecam1 double-positive cells from aHDFs, involving transduction of 3 key factors, treatment with 2 inhibitors of epithelial-mesenchymal transition (siTWIST1 and rosiglitazone), and additional incubation of sorted VE-cadherin+ cells for 2 weeks (Figure 3). The resulting double-positive cells demonstrated similar genetic profiles to those of human umbilical vein ECs and gastroepiploic artery ECs. These human iECs also showed authentic endothelial characteristics, including morphology and function (Figure 4).

Figure 3.

Schematic of the protocol to convert adult human dermal fibroblasts (aHDF) to induced endothelial cells (iECs) that are double-positive for VE-cadherin (VE-cad) and PECAM-1.

Figure 4.

Endothelial characteristics of iECs. (A) Optical microscopic findings showing a cobblestone appearance. Scale bar=250 μm. (B) Scanning electron microscopic findings showing an endothelial monolayer. Scale bar=10 μm. (C) In vitro capillary tube formation on Matrigel. Scale bar=250 μm. HUVEC, human umbilical vein endothelial cell.

Other groups have also reported direct conversion into ECs in humans. The details of each protocol are presented in Table 2. Ginsberg et al reported that the constitutive co-expression of ERG1 and FLI1 combined with transient ER71 expression reprogrammed amniotic cells into ECs.⁵² In their protocol, approximately 60% of the cells expressed VE-cadherin/Pecam1 double-positivity after a 4-week-derivation process. Notably, EC-specific genes were only minimally induced in adult fibroblasts by their combination of transcription factors. Wong et al showed that transduction with a combination of ER71, FLI1, GATA2, and KLF4 converted human neonatal fibroblasts into ECs.⁵³ Approximately 16% of the 4-factor-transduced cells expressed Pecam1. However, flow cytometry revealed that these cells expressed only dim fluorescence for Pecam1. In addition, the converted ECs were only characterized using acetylated low-density lipoprotein incorporation and Matrigel tube formation. Furthermore global gene expression analysis was not performed. Lee et al screened 7 transcription factors and found that transduction with ER71 alone best induced the endothelial features in human neonatal fibroblasts.⁵⁴ The derivation protocol spanning 3 months included cell sorting based on Vegfr2 expression on day 7. Notably, Vegfr2, which was used as a readout, is a mesodermal marker that is not very specific to ECs. Morita et al reported that transduction with ER71 alone directly converted human adult fibroblasts into ECs;⁵⁵ however, endothelial NO synthase, an essential characteristic of ECs, was not expressed in their converted ECs. Further, we could not derive VE-cadherin/Pecam1 double-positive cells following their protocol in our settings. Overall, we confirmed that our iEC protocol was the most effective for directly converting adult fibroblasts into fully functional ECs in humans.⁴²

Table 2. Comparison of Different Endothelial Conversion Protocols

Original cells	Conversion factors	Culture media	Readout method	Reference
Human adult dermal fibroblasts	ER71, KLF2, TAL1, siTWIST1	(Day0~28) EGM-2MV + rosiglitazone	VE-cad/PECAM1(+/+) at day42	Han et al (2021)42
		(~Day29) EGM-2MV
Amniotic cells	ER71, FLI1, ERG	Medium 199 + EC suppl + heparin + SB431542	VE-Cad(+) at day21	Ginsberg et al (2012)52
Human neonatal dermal fibroblasts	ER71, GATA2, FLI1, KLF4	(Day0~3) DMEM + BMP4 + VEGF + bFGF	PECAM1(+) at day14	Wong et al (2016)53
		(~Day4) EGM-2MV+SB431542
Human neonatal dermal fibroblasts	ER71	(Day0~7) DMEM	KDR(+) at day7	Lee et al (2017)54
		(~Day8) EGM-2 + VEGFA
Human embryonic lung fibroblasts, human adult dermal fibroblasts	ER71	EGM-2 + VEGF + bFGF	PECAM1(+) at day15	Morita et al (2015)55

Completeness of Direct Conversion

The induction of complete conversion is an essential subject in the field of direct cell conversion. CellNet, a network biology platform assessing the fidelity of cell fate conversions, suggested that the main deficiencies of direct conversion included failure to adequately silence donor cell gene regulatory networks (GRNs) and expression of aberrant GRNs, resulting in hybrid cell types.⁵⁶ CellNet revealed that induced hepatocytes (iHep), which were claimed to be directly converted to hepatocytes by transduction of 2 transcription factors,⁵⁷ harbored considerable hindgut identity.⁵⁸ Morris et al found long-term functional colon engraftment of iHeps, indicating they actually represent endodermal progenitor cells.⁵⁸ Regarding iCM, although ≈25% of mouse fibroblasts were reported to be converted into α-myosin heavy chain expressing cells following an iCM derivation protocol in vitro,¹⁵ among them only 30% expressed cardiac troponin T, a specific sarcomeric marker of differentiated mature CMs. Furthermore, the percentage of fibroblasts completely reprogrammed to beating CMs in vitro was rather small, but many more were partially reprogrammed.⁵⁹

Our study also showed that VE-cadherin single-positivity was not sufficient to define fully reprogrammed iECs, whereas VE-cadherin/Pecam1 double-positivity represents complete conversion to the endothelial lineage.⁴² Regarding iPSCs, several criteria to confirm the fully reprogrammed status are well established, including (1) morphology identical to ESCs, and unlimited self-renewal; (2) genetic and epigenetic profiles closely similar to those of ESCs, and expression of key pluripotency factors and surface antigens specific to ESCs; (3) independence from transgene expression; and (4) able to differentiate into lineages of all 3 germ layers, which can be demonstrated by in vitro differentiation, teratoma formation, chimera contribution, germline transmission, or tetraploid complementation in the order of increasing stringency.⁶⁰ In contrast, there is no consensus on how to define the completeness of direct cell fate conversion. Based on our study, we propose the following criteria to confirm full reprogramming into ECs. (1) Morphological attributes: optical microscopy showing typical endothelial cobblestone appearance, scanning electron microscopy demonstrating an endothelial monolayer, and transmission electron microscopy showing a Weibel-Palade body specific to ECs; (2) genetic and epigenetic profiles indistinguishable from ECs: whole transcriptome ribonucleic acid (RNA) sequencing, CpG island methylation patterns, double-positivity for VE-cadherin and PECAM1, and downregulation of the original cell-specific (usually fibroblast-specific) genes; (3) silencing of exogenous gene expression; and (4) multiple endothelial functional assays, including Matrigel tube formation or 3D in vitro angiogenesis, NO production, acetylated low-density lipoprotein uptake, anticoagulation assays on monolayered cells, permeability assays, and in vivo angiogenesis assays, such as a hindlimb ischemia model. These criteria will help researchers to correctly define converted ECs and standardize the derivation methodology for iECs.

Unique Advantage of Direct Conversion Compared With Dedifferentiation

One of the most important merits of direct conversion is that it does not require induction of pluripotency. Single-cell RNA sequencing demonstrated that during directed differentiation of iPSCs into ECs, various heterogeneous cell types, including immature CMs, hepatic-like cells, and vascular smooth muscle cells, were induced.⁶¹ Considering the nature of pluripotency, this kind of heterogenic differentiation is unavoidable. Another single-cell RNA sequencing study revealed that after following one of the most rapid and efficient protocols to derive ECs from pluripotent stem cells,⁶² >4% of the derived cells still expressed stage-specific embryonic antigen 4 (SSEA4), a pluripotency marker, and ≈0.04% of the cells co-expressed a combination of pluripotency markers, SSEA4/transcription-associated protein-1-81 (TRA-1-81).⁶³ Considering the unlimited self-renewal potential of pluripotent stem cells, even a small proportion of cells expressing pluripotency markers cannot be neglected, and may make clinicians and patients hostile to iPSC-derived cells for therapeutic purposes.

Another essential merit of direct conversion, compared with the iPSC technique, is that direct conversion can be induced at the target tissue or organ in situ. Fibroblasts are one of the most abundant cell types in the body. For example, CMs only account for 30–40% of cell numbers in the heart, and the majority of the remaining cells are cardiac fibroblasts.⁶⁴ Thus, fibroblasts in the diseased organ in situ can be used as a good source for direct conversion into the desired cell type. Accordingly, the iEC conversion technique can be utilized in 2 different ways (Figure 5): (1) for cell therapy, SFBs of the patient are harvested and directly converted into iECs, and these iECs are then transplanted into the target tissue or organ to relieve ischemia; (2) for gene therapy, 3 key iEC factors (Er71, Klf2, and Tal1) are directly introduced into fibroblasts of the target tissue or organ via non-viral or viral vectors. Subsequently, the transfected fibroblasts were converted into iECs, resulting in new vessel formation in situ. For example, a poly-cistronic lentivirus encoding all 3 iEC factors can be transduced to the myocardium of a patient with myocardial infarction or the limb muscles of a patient with critical limb ischemia to salvage ischemic tissue.

Figure 5.

Two different strategies for regenerative therapy based on induced endothelial cell (iEC) technology for ischemic cardiocerebrovascular diseases. (A) Ex vivo converted iECs can be transplanted to the target tissue/organ under ischemia, or (B) direct reprogramming into iECs can be induced in vivo. These strategies can be adopted, for example, to form new vessels in the ischemic heart of patients suffering from myocardial infarction.

To date, in vivo direct conversion has been demonstrated in several cell types, such as CMs,^59,6566 neurons,^67–69 endocrine β-cells,¹³ and hepatocytes.⁷⁰ Notably, in vivo iCMs were found to be more fully reprogrammed and more closely resembled authentic CMs compared with in vitro reprogrammed iCMs.^56,59 It was speculated that the native microenvironment (e.g., extracellular matrix, secreted proteins, and tissue stiffness) may provide a better milieu for enhanced direct conversion.⁵⁹ In vivo delivery of cardiac reprogramming factors into infarcted hearts significantly improved cardiac functions, and attenuated the extent of fibrosis and scar size in a murine myocardial infarct model.^59,66 Conversion of cardiac fibroblasts to CMs may directly reduce fibrosis and scar formation, and, in addition, the behavior of fibroblasts infected by reprogramming factors may be altered and may show impaired function to induce fibrosis.⁷¹

Transplantation of iPSC- or ESC-derived CMs into infarcted myocardium was reported to induce ventricular tachycardia.^72,73 Given that ventricular arrhythmias can be potentially life-threatening, this finding may impede the clinical application of iPSC- or ESC-derived CMs. One study reported that iCM conversion in vivo was not associated with the development of arrhythmias.⁵⁹ However, more research is warranted on this issue. Theoretically, iEC conversion in vivo should not induce arrhythmogenicity, because the converted ECs do not have spontaneous electrical activity. Thus, gene therapy for in vivo direct conversion to iECs would be advantageous to improve post-infarct myocardial function by increasing blood supply compared with other strategies, but this hypothesis needs to be verified by future studies.

Challenges and Future Directions

One important limitation of direct conversion technology is that it does not rejuvenate cells through the lineage reprogramming process.⁷⁴ iPSC generation resets the telomere size, mitochondrial metabolism, gene expression profile, and remodels epigenetic marks, leading to cellular rejuvenation.⁷⁵ In contrast, directly reprogrammed cells retain their cellular age.⁷⁶ Thus, direct endothelial conversion of fibroblasts from older patients results in old iECs. This may limit the therapeutic efficacy of direct reprogramming, as old cells are thought to have low proliferation capacity and restricted cellular functions, and most candidates for cell/gene therapy would have age-related diseases such as ischemic cardiocerebrovascular disease and heart failure. In addition, aging or senescence per se acts as a barrier for nuclear reprogramming, thus limiting its efficiency.⁷⁷ To overcome these aging or senescence-related limitations, modulation of cell cycle regulators has been attempted. Senescence is associated with the upregulation of p16 and p19, and activation of p53.⁷⁸ In this context, repression of p16 and p19, or overexpression of human telomerase reverse transcriptase was shown to enhance neuronal lineage conversion from fibroblasts,^29,78 whereas expression of p53 blocks neuronal conversion.⁷⁸ Interestingly, overexpression of cyclin-dependent kinase 1 (CDK1), CDK4, cyclin B1, and cyclin D1 was found to induce cell division in post-mitotic CMs.⁷⁹ This other type of reprogramming to induce forced replication can be adopted as a strategy to overcome aging/senescence-associated challenges in direct lineage conversion. However, caution should be exercised when utilizing this strategy, because regulation of the cell cycle is essential to maintain genomic integrity and avoid tumorigenesis.

The gene delivery methodology is another challenge that needs to be overcome. Currently, retroviruses or lentiviruses are widely used in direct conversion for research purposes, because long-term stable expression of target genes is easily achieved using these viruses. In particular, lentiviral vectors can infect non-dividing cells, resulting in higher transduction efficiency.⁸⁰ However, retroviruses and lentiviruses integrate their genetic material into the host genome.⁸⁰ This unique characteristic can cause insertional mutagenesis associated with random integration, which may preclude the wide adoption of this delivery method for therapeutic purposes. Alternatively, adenoviruses, adeno-associated viruses, or Sendai virus without integration properties can be used. However, these viruses induce transient expression of the delivered genes, and thus reprogramming efficiency is relatively lower than that of genome-integrating viruses.^81,82 Further, viral vectors commonly have issues associated with cytotoxicity, immune response, and cargo capacity.⁸³ Plasmid DNA or RNA delivered by non-viral carriers such as liposomes and polymers is free from most safety issues of viral delivery. However, the low transfection efficiency and gene expression levels are major limitations. Recently, nanotechnology such as the use of lipid nanoparticles has drawn researchers’ interest, as it provides a non-viral gene carrier resembling the features of viral vectors, but does not pose the related safety concerns.⁸⁴

Conclusions

The discoveries and advancements in nuclear reprogramming clearly demonstrate that cell fate is not fixed, but rather is flexible. Transduction of only a few key transcription factors can reconstruct the transcriptional network and directly convert one cell type into another. This technology holds great practical promise because it does not involve pluripotency induction, and can thus avoid tumorigenesis risk. Further technical improvements to circumvent cellular senescence-related issues and obtain a safe and efficient gene delivery tool are therefore warranted to realize cell/gene therapy based on direct cell conversion.

Disclosure

This manuscript was supported by a grant funded by the Korean Ministry of Health and Welfare (HI14C1277, HI 17C 2085).

Supplementary Files

Please find supplementary file(s);

https://doi.org/10.1253/circj.CJ-21-0703

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