Evolving principles underlying neural lineage conversion and their relevance for biomedical translation

Scientific and technological advances of the past decade have shed light on the mechanisms underlying cell fate acquisition, including its transcriptional and epigenetic regulation during embryonic development. This knowledge has enabled us to purposefully engineer cell fates in vitro by manipulating expression levels of lineage-instructing transcription factors. Here, we review the state of the art in the cell programming field with a focus on the derivation of neural cells. We reflect on what we know about the mechanisms underlying fate changes in general and on the degree of epigenetic remodeling conveyed by the distinct reprogramming and direct conversion strategies available. Moreover, we discuss the implications of residual epigenetic memory for biomedical applications such as disease modeling and neuroregeneration. Finally, we cover recent developments approaching cell fate conversion in the living brain and define questions which need to be addressed before cell programming can become an integral part of translational medicine.


An introduction to cell programming and lineage conversion
The term cellular programming describes the modulation of transcriptional networks underlying cell identity. Research on cell programming has a remarkable history. First reports describing the principal feasibility of converting one cell type into another were published as early as 1987, when Davis, Weintraub, and Lassar derived myoblasts by overexpressing the myoblast transcription factor (TF) Myod3 in a mouse fibroblast line 1 . However, at that time, cellular programming was restricted to the conversion of lineage-related cells of the same germ layer (see the 2009 review by Graf and Enver 2 ). This changed dramatically when Kazutoshi Takahashi and Shinya Yamanaka revealed that overexpression of the four TFs Oct3/4, Sox2, Klf4, and c-Myc is sufficient to induce a pluripotent state in mouse 3 and human 4 fibroblasts (for a more detailed review, see 5). Today, protocols are available to obtain these embryonic stem cell (ESC)-like induced pluripotent stem cells (iPSCs) from various species and starting cell types.
The success of the iPSC approach demonstrated that cell programming is not restricted to the conversion of related cell types and fueled attempts to achieve somatic-to-somatic cell conversion across germ layers. One avenue pursued in this direction has been the combination of time-restricted expression of the classic iPSC reprogramming TF cocktails with growth factors and small molecules promoting neural lineage development. An exemplar for such a "partial" reprogramming is the Oct3/4-, Sox2-, Klf4-, and c-Myc-driven derivation of neural stem cells (NSCs) from fibroblasts 6-10 or blood cells 11 , where transgene expression was combined with an exposure to FGF2, FGF4, and/or EGF 6,7,10,11 , FGF2 and/or EGF in conjunction with LIF 8 , or LIF in combination with the TGFβ-inhibitor SB431542 and the GSK3β-inhibitor CHIR99021 9 . It is worth mentioning that in accordance with the transient expression of TFs used for generating iPSCs, partial reprogramming to NSCs may involve a short transit through a pluripotency-like state and can result in mixed cultures of iPSCs and NSCs 12,13 . Interestingly, such a pluripotency transit can also occur without forced Oct4 expression. Lineage tracing using an Oct4 reporter revealed that NSCs derived by overexpression of Sox2, Klf4, and c-Myc in conjunction with the neural-specific TF Brn4 instead of Oct4-a protocol originally published by Han et al. 14 in 2012-originate from Oct4-expressing iPSC-like cells 12 . Such a pluripotency transit can be used to mechanistically discriminate partial reprogramming from direct cell fate conversion (hereafter also denoted as transdifferentiation) based on the overexpression of lineage-specific TFs.
A major breakthrough concerning transdifferentiation across germ layers was in 2010, when the group of Marius Wernig succeeded in inducing neurons from mouse fibroblasts by overexpressing the neural lineage-specific TFs Ascl1, Brn2, and Myt1l 15 . Soon thereafter, conversion of human fibroblasts to induced neurons (iNs) was achieved by using exactly this ASCL1, BRN2, and MYT1L TF combination 16,17 ; ASCL1, BRN2, and MYT1L in conjunction with NEUROD1 18 ; or BRN2 and MYT1L together with the neuronal microRNA miR124 19 (Table 1).
While direct transdifferentiation into a neuron remains a fascinating concept, the applicability of this approach can be limited by the fact that neurons are post-mitotic, thereby restricting largescale applications. In addition, since not all cells undergo successful transdifferentiation, elimination of partially reprogrammed cells remains an issue. Finally, each transdifferentiated neuron represents a singular event and thus cannot be subjected to common batch control-based quality-control regimens, limiting the degree of standardization that can be reached with iN cultures. In light of this, expandable NSCs or neural progenitor cells (NPCs) could offer an interesting alternative. Indeed, several groups reported on the successful transdifferentiation of mouse 20,21 and human 22-27 fibroblasts into still-proliferative NSCs or NPCs using different NSC-enriched TFs or TF combinations (Table 2). Subsequently, other somatic cells, too, were found to be amenable to direct neural conversion. In this context, easily accessible cell populations such as blood-derived 28-31 and urine-derived 32 cells are of particular interest. Alongside converting non-central nervous system (non-CNS)-resident cells, there has been significant progress with neural conversion of non-neural, CNS-resident cells such as brain pericytes 33,34 and yolk sac-born microglia 35 , which both represent attractive candidates for in vivo reprogramming. In parallel, transdifferentiation of astrocytes-which can be regarded as derivatives of neurogenic radial glia cells at the end of neural development (for further details on the relationship of radial glia cells, NSCs, and neurogenesis, see Falk and Götz 36 )-has been rapidly developing 37-39 , although most studies have been focusing on transdifferentiating astrocytes in vivo (see "Destabilizing and converting cell fates in vivo" section below and Table 3). As for the in vitro conversion of astrocytes into neurons, Benedikt Berninger, Magdalena Götz, and colleagues already showed in 2007 that this can be achieved by overexpression of the single neurogenic TF Ngn2 or Ascl1 40 . In the meantime, additional paradigms based on TF combinations 41 , microRNAs 42 , or small molecules 43 (or a combination of these) have been reported for astrocyte-to-neuron conversion.
Since the beginning of this century, the cell programming toolbox has expanded rapidly. The aims of this review are to concisely recapitulate recent advances in this field, to briefly sum up our current understanding of general mechanisms underlying cell fate conversion, to summarize commonalities and differences between the available methods, and to discuss their pros and cons with respect to biomedical applications such as disease modeling and neuroregeneration.

Boosting transdifferentiation efficiency and finetuning sublineage specification
The first seminal reports on transdifferentiating somatic cells into neurons raised strong interest to make this process more efficient and, in particular, to tailor it toward the generation of distinct neural subpopulations. Since neurons are generally post-mitotic, conversion efficiency is a major limiting factor. Several studies addressed this bottleneck. It has been shown that modulation of signaling pathways by small molecules significantly improves iN conversion. For example, combined inhibition of SMAD and GSK3 signaling in human fibroblasts by small molecules can increase iN purity and yield (percentage of neurons in relation to  switch from glycolysis to oxidative phosphorylation) efficiently improves human fibroblast-to-neuron conversion 62 . Moreover, in inducible viral systems, delivering multiple programming factors by all-in-one, polycistronic vectors 62 and including a recovery phase between viral transduction and transgene activation 54 have been shown to increase conversion efficiency. Lastly, reducing reprogramming barriers in somatic cells, such as inhibiting REST signaling in human fibroblasts 60 , overcoming senescence 55 , or inducing epigenetic remodeling by TET1 activation 70 , has been reported to boost iN generation, too.
In addition to increasing iN conversion efficiency as such, the generation of defined neuronal subpopulations has been a key focus of this emerging field. While the initial TF combinations used for iN generation resulted primarily in excitatory neurons, these cultures also contained inhibitory GABAergic neurons 18,19,35,47,52 . However, some groups reported on iN paradigms that strongly enrich for either glutamatergic 63-65 or GABAergic 33,34,66,67 neurons. With respect to potential clinical prospects, the controlled induction of midbrain dopamine neurons and striatal medium spiny neurons (MSNs)-the prime targets of Parkinson's disease (PD) and Huntington's disease (HD), respectively-remains a key focus. In order to derive dopaminergic iNs from human fibroblasts, the classic iN reprogramming cocktail of ASCL1, BRN2, and MYT1L can be combined with the dopaminergic fate-specifying TFs LMX1A and FOXA2 17 or a further enriched combination of LMX1A, LMX1B, FOXA2, and OTX2 50,54 . Alternatively, Ascl1 alone has been shown to be sufficient to induce a dopaminergic fate in fibroblasts when combined with Nurr1 and Lmx1a 69 ; Nurr1, Lmx1a, Foxa2, Pitx3, and En1 68 ; or Nurr1, Lmx1a, and miR124 70 . For the derivation of MSNs, combined overexpression of the CNS-enriched miR9/9* and miR124 with MYT1L and the striatal TFs CTIP2, DLX1, and DLX2 was used to convert human fibroblasts into mainly DARPP32positive GABAergic neurons 71 . Direct conversion has also been used to generate serotonergic neurons 73,74 as well as peripheral sensory neurons 80,81 and motor neurons (MNs) 75-78 (see Table 1 for further details). A number of these directly converted neuronal subpopulations have been successfully used for in vitro disease modeling 72,77,81 and drug testing 77 (for the use of iNs in disease modeling, see also Drouin-Ouellet et al. 113 ). In parallel to improving trans-germ layer conversion, the generation of neuronal subtypes from astrocytes has been refined. Pioneering studies by Berninger and Götz already indicated that overexpression of Ngn2 yields mostly glutamatergic neurons whilst direct conversion of astrocytes with Dlx2 results in neurons biased toward a GABAergic phenotype 82,114 . More recently, in vivo transdifferentiation into neurons with a predominantly dopaminergic fate was achieved 84 .
With regards to fine-tuning direct conversion paradigms, it is worth mentioning that experimental tools other than classic retroviral or lentiviral systems have been employed for the delivery of TFs or activation of endogenous reprogramming-inducing genes. These include non-integrating viruses 30 110 Rouaux and Arlotta (2013)

Forward programming as fallout of transcription factor-based somatic cell fate conversion
Given the tremendous efficacy of TFs in converting somatic cell fates, it is not surprising that this concept has been rapidly adopted to instruct cell fates from pluripotent stem cells (PSCs), thereby replacing or supplementing classic differentiation paradigms using extrinsic factors. "Forward programming" approaches such as the overexpression of neurogenins (NGNs) [115][116][117][118][119][120]121 in human PSCs significantly accelerate neuronal differentiation and maturation times. These PSC-derived human neurons have been shown to become electrophysiologically functional as early as two weeks after NGN induction 115,116 , and synchronized network activity can be detected already after three weeks in culture 122 . This acceleration is associated with an increased synchronization of the differentiation process, which facilitates disease modeling applications focusing on functional phenotypes as, for example, in schizophrenia and autism 123,124 and tuberous sclerosis and epilepsy 115,117 . As with somatic cell fate conversion, combined overexpression of classic neurogenic TFs with TFs promoting distinct regional fates can be used to further fine-tune the generation of distinct neuronal subtypes. Notably, the effect of a given pioneer TF in PSCs might be quite different from that in somatic cells. This was demonstrated in 2016 by Smith et al., who studied the effect of NGN2 overexpression in human fetal fibroblasts 59 . They revealed that although NGN2 is able to act as a pioneer TF in this transdifferentiation setting (that is, targeting regions in a closed chromatin state), converting fibroblasts into iNs with NGN2 alone is extremely inefficient. However, this low efficiency is significantly enhanced by the small molecules forskolin and dorsomorphin, which promote chromatin accessibility at pro-neural NGN2 binding sites. More specifically, forskolin and dorsomorphin enhance the enrichment of CREB1 at sites bound by NGN2, thereby inducing the expression of the pro-neural gene SOX4. SOX4, in turn, elicits further downstream chromatin remodeling and consequently facilitates the activation of other pro-neural genes such as NEUROD1 and NEUROD4 59 . For other somatic cell types, different pioneer factors might be required to promote cell fate conversion. In mouse microglia, for instance, not Ngn2 or Ascl1 but Neurod1 acts as a neuronal pioneer TF, specifically inducing transcription of its bivalently marked pro-neural target genes 35 . Along the same lines, oligodendrocytes, which also feature bivalent histone modifications at pro-neural Neurod1 target genes, were successfully reprogrammed into neurons by Neurod1 overexpression 35 .
As the reprogramming field progressed, major advances were made in profiling cell fate trajectories by single-cell RNA sequencing (scRNAseq). Using this technology, Treutlein et al. studied the conversion of mouse embryonic fibroblasts to iNs and specifically analyzed the contribution of the neuronal pioneer TF Ascl1 to the induction and stabilization of the fibroblast-to-iN fate switch in the Ascl1-Brn2-Myt1l paradigm 129 . Concordant with the results of Wernig's group, they showed that overexpression of Ascl1 alone is sufficient to homogenously induce downregulation of fibroblast-enriched transcriptomic signatures, to upregulate the expression of neuronal genes, and to promote cell cycle exit. They found the continued expression of Ascl1 as well as co-expression of Brn2 and Myt1l to be essential for the stabilization of neuronal fate and subsequent neuronal maturation, whereas silencing of Ascl1 in the course of the conversion process resulted in the reappearance of fibroblast signatures 129 . Notably, the majority of Ascl1-only-induced cells do not acquire a neuronal identity, even if Ascl1 expression levels are maintained, but activate a myocyte-related transcriptional program 129 . This observation might be explained by the lack of Myt1l induction in Ascl1-only conditions. Mall et al. investigated the role of this non-pioneer TF during fibroblast-to-neuron conversion and revealed that its main function is to interact with the Sin3b-HDAC1 complex to repress non-neuronal transcriptional programs 130 . Myt1l-repressed targets include genes promoting proliferation, such as Hes1, and genes inducing alternative lineages, including targets relevant for myocyte differentiation 130 . Together, these data indicate that, in addition to pioneer factors, secondary fate-specifying or alternative fate-repressing cues (or both) are necessary to ensure proper phenotype stabilization. Consequently, Tsunemoto et al. recently screened a library of 598 TF pairs for their ability to convert mouse embryonic fibroblasts into functional iNs 49 . As expected, almost all successful combinations included at least one member of the Ascl, Ngn, or Neurod families. However, pairs of pro-neural TFs comprising no pioneer TF also yielded functional iNs, demonstrating that pioneer TFs are not an indispensable condition for direct cell fate conversion 49 . Along similar lines, the group of Lei Qi performed a CRISPR activation screen to identify single TFs and TF combinations that promote differentiation of mouse ESCs and direct conversion of fibroblasts into neurons 48 . In addition to known pro-neural TFs such as Ngns or Brn2, their top hits included non-pioneer TFs and even non-neural-specific TFs such as the epigenetic regulator Ezh2 48 .
Taken together, the currently available data support a two-stage architecture of the conversion process. First, target cell typespecific genes need to be made accessible in case they are in an unfavorable chromatin state in the starting cell type. In addition to pioneer TFs, epigenetic modifiers or other factors modulating chromatin accessibility can exert this effect. Overexpression of miR9/9* and miR124, for instance, has been shown to promote gradual remodeling of chromatin accessibility at fibroblastspecific enhancers (change to closed chromatin) and chromatin opening at pan-neuronal gene loci 78 . Second, after induction of epigenetic plasticity, acquisition and stabilization of a new cell fate have to take place. Although this process can be initiated and orchestrated by pioneer TFs too, it mostly involves additional TFs. These can be co-transduced in the starting cell along with the pioneer TF (that is, by overexpressing TF combinations) or induced by small molecules used to promote the direct conversion process or they are direct transcriptional targets of the pioneer TF and thus secondarily induced by the pioneer itself. Eventually, pioneer as well as non-pioneer TFs instruct the adoption of a specific cell fate through either active induction of target lineage-specific genes (as was demonstrated for, for example, Ascl1 129 or Neurod1 35 ) or transcriptional repression of genes instructing alternative cell fates (as shown for, for example, Myt1l 130 ). Notably, however, the process of fate acquisition might involve additional intermediate steps, since scRNAseq time-course analyses of the iN conversion process indicate the presence of transient, unstable progenitor-like identities before a stable neuronal phenotype is adopted 34,129 . As overarching mechanistic principles underlying cell fate conversion become increasingly uncovered, it is important to note that the exact mechanisms of fate switches will always comprise components highly specific to the identity of the interconverted cell types and the individual conversion paradigm.

Tampering with epigenetic age
The epigenetic memory of a cell falls into two major categories: cell fate and age. Since significant transcriptomic and epigenetic remodeling plays a pivotal role in the process of cell programming, it seems natural to ask how different programming paradigms affect a cell's aging signature. However, age is a highly multifaceted phenomenon and hard to assess by simple means (see the 2015 review by Studer, Vera, and Cornacchia 131 ). Some aspects of cellular aging, such as compromised nuclear architecture, cannot be easily assessed in a quantitative manner. Others, such as telomere length, might not strictly correlate with biological age, depending on the tissue context 132 . One alternative way to estimate the biological age of a cell independent of its somatic cell fate is to analyze DNA methylation (DNAm) signatures and apply algorithms calculating a DNAm age 133 . When applied to iPSC generation, DNAm ages have been shown to be reset upon induction of pluripotency, which is in line with the fact that iPSC reprogramming resets the starting cell's identity back to an embryoniclike state 133,134 . Thus, somatic-to-iPSC reprogramming represents a tool to derive epigenetically rejuvenated cells. Conversely, in 2015, the groups of Yixuan Wang and Fred Gage demonstrated that aging hallmarks such as age-specific transcriptional signatures and the age-dependent loss of nucleocytoplasmic compartmentalization are preserved in mouse 135 and human 56 iNs, respectively. One year after these reports, it was demonstrated that the DNAm ages of iNs are retained, too, and almost perfectly correlate with their donors' chronological ages 57 . Over the last two years, several other studies corroborated the notion that ageassociated cellular alterations such as senescence, susceptibility to DNA damage, mitochondrial defects, loss of heterochromatin, and alterations in nuclear organization are preserved in fibroblast-derived iNs 61,79 . These findings indicate that iNs maintain not only epigenetic but also functional age-related phenotypes of their cells of origin. Interestingly, all of these studies were conducted in a scenario where the converted cells immediately enter the postmitotic stage of an iN. Thus, we became interested in the question of how age preservation would work upon transdifferentiation into a proliferative somatic stem cell population. Following up on this idea, we used temporary overexpression of SOX2 and c-MYC to convert adult peripheral blood cells into induced NSCs (iNSCs). Using the Horvath and other epigenetic age predictor algorithms, we found that iNSCs generated in this manner undergo massive epigenetic rejuvenation similar to what is observed during iPSC reprogramming 30 . This observation is noteworthy, as our conversion approach is OCT4-free and there is no evidence of a transit through a pluripotent state. Although the mechanism underlying the reset of biological age remains to be unveiled, this finding strongly suggests that epigenetic rejuvenation does not require an intermediate pluripotent stage and can also be achieved during transdifferentiation of somatic cell types. This notion is further supported by very recent analyses of DNAm changes upon iPSC reprogramming, which show that the reset of DNAm age and the establishment of a stable and self-sustaining pluripotent state follow different time dynamics 136 . From a conceptual point of view, these observations support the idea that epigenetic rejuvenation, in principle, can be achieved in somatic cells.
The question of whether or not reprogrammed cells preserve age signatures is especially relevant when it comes to modeling age-related diseases. In particular, successful modeling of neurodegenerative diseases might depend on the preservation of cellular defects naturally accumulating over an organism's life span. The importance of age preservation for disease modeling was recently illustrated in the context of HD. The group of Andrew Yoo found that aggregation of the disease-causing mutant huntingtin protein can be readily recapitulated in directly converted MSNs but not in iPSC-derived MSNs, a phenomenon the authors attributed to the erasure of age signatures such as the restoration of proteasomal activity in iPSC-derived MSNs 72 . Acknowledging that the lack of aging hallmarks in iPSC-derived somatic cells can impede modeling of age-associated pathophenotypes, strategies such as progerin overexpression or telomerase inhibition have been explored to promote the emergence of age-associated phenotypes 137,138 . Owing to their age memory, directly converted neurons might not require additional age-promoting treatments for modeling late-onset neurodegenerative diseases, for example. However, it is fair to say that iNs might, vice versa, be less suitable for modeling neurodevelopmental disorders.

Somatic memory and authenticity
Although some diseases affect neurons rather broadly, others are known to target preferentially specific subtypes such as PD, which is associated with a loss of mesencephalic dopaminergic neurons in the substantia nigra. Since cellular pathomechanisms might be cell type dependent, the authenticity of the transdifferentiated neural subpopulation might contribute significantly to the validity and power of cellular disease models. While there is evidence for low levels of residual somatic memory in low-passage iPSCs 134,139,140 , these signatures appear to vanish after prolonged in vitro cultivation 140 . This presents differently in directly converted cells. Tsunemoto et al. analyzed four fibroblast-derived iN populations reprogrammed by different TF combinations and revealed that although the global transcriptome of iNs is highly similar to that of endogenous neurons, all iN populations showed residual low-level expression of a subset of fibroblast-specific genes 49 . Residual somatic signatures were also recently reported for iNSCs. Thier et al. derived iNSCs with neural plate border identity from different populations of human fibroblasts and blood cells 31 . They found that dermal fibroblast-derived but not blood-derived iNSCs still express the fibroblast marker COL3A1, although other fibroblast-lineage markers are significantly downregulated upon transdifferentiation 31 . Nevertheless, residual somatic signatures in directly converted cells appear to be insufficient to maintain the identity and function of the cell of origin. For example, hepatocyte-derived iNs were shown to retain more than 10% of the liver-specific transcriptomic signature, but resulting iNs are capable of neither secreting albumin nor producing urea 44 .
However, the questions of whether and to what extent the function of the converted iNs can be compromised by residual somatic signatures of the donor cell certainly merit further investigation, and recent data suggest that authenticity is an issue not restricted to direct cell fate conversion. Ichida et al. compared primary mouse spinal MNs with ESC-derived, iPSC-derived, and directly converted MNs and revealed that all in vitro-derived MN populations, regardless of the reprogramming paradigm used, expressed only about 55% to 86% of the primary MN transcriptome 75 . These differences were accompanied by even more pronounced discrepancies in the methylation status 75 . From a technical point of view, this study illustrates a fundamental bottleneck of contemporary cell fate reprogramming and conversion research: traditionally, cell fate identification has been based mostly on the expression of cell type-specific marker profiles, and, if applicable, further characteristic features such as specific functional properties, including electrophysiological data, have been considered. These approaches are biased, however, as they are hypothesis driven. More holistic approaches such as the in-depth analysis of transcriptomic data, ideally in single-cell resolution, and comparative methylation analysis, as performed by the Eggan lab, might represent means to provide more reliable and biologically meaningful measures of cell identity and authenticity. From a biological perspective, such findings also point to more general limitations of in vitro cell systems in recapitulating in vivo scenarios. On the other hand, it remains unclear what degree of somatic authenticity is eventually required to, for example, recapitulate disease-specific phenotypes-an issue which also depends on the specific experimental hypothesis. For other biomedical applications such as replacement of distinct neuronal subpopulations, utmost authenticity will always represent the ultimate goal.

Destabilizing and converting cell fates in vivo
Translation of in vitro paradigms of direct cell fate conversion to an in vivo scenario remains one of the most fascinating perspectives of regeneration. From a translational point of view, such approaches could eventually replace cell transplantation. From a biological perspective, transdifferentiation of region-specific cells in a native tissue environment might represent the ultimate approach to approximate authenticity.
In the CNS, the longest history of in situ transdifferentiation has astrocyte-to-neuron conversion (Table 3), starting with the observation that antagonizing Olig2 or overexpressing Pax6 after traumatic brain injury enables neurogenesis from resident reactive astrocytes 37 ; this phenomenon has also been recapitulated after infliction of mild brain ischemia 38 . Similarly, Neurod1 has been shown to convert reactive astrocytes as well as NG2positive progenitors into neurons in mouse stab injury and Alzheimer's disease models 108

Challenges for clinical application
Even though in vivo conversion is a promising strategy to exploit endogenous sources for cell replacement, a number of limitations have to be overcome before this approach is fit for clinical translation. First, the delivery of fate-instructing factors has to be good manufacturing practice compliant and applicable in living humans. Here, established viral vector systems successfully applied in gene therapy approaches such as adenoassociated viruses 104,107,112 might represent an attractive solution. Alternatively, non-viral approaches such as transducible proteins, mRNAs, or small molecules might qualify for delivering the required cell programming cues in vivo (for a review on recent technologies facilitating in vivo reprogramming, see Larouche and Aguilar 146 ). Whatever delivery system is chosen, it has to enable factor distribution to the lesion site. For focal lesions, stereotaxic delivery can be considered, but more global cell loss might require modes of systemic administration that are not impeded by the blood-brain barrier. Moreover, dependent on the individual transdifferentiation regimen, multiple rounds of factor administration or delivery of depots such as scaffoldbound conversion factors might be necessary (reviewed in 2019 by Larouche and Aguilar 146 and Bruggeman et al. 147 ).
In vivo conversion is further complicated by the fact that this process can be strongly context dependent. Grande et al. showed that Ngn2-mediated conversion of proliferating non-neuronal cells yields GABAergic neurons in the mouse striatum but results in the emergence of glutamatergic neurons in the neocortex 100 . In addition, the local microenvironment can affect conversion efficiency. Wang et al. 106 found that decreasing p53-p21 signaling increases the yield of astrocyte-derived neuroblasts by preventing p53-induced cell cycle exit, while locally secreted neurotrophins can support their maturation 105 . Götz and her team reported that counteracting oxidative stress and ferroptosis can significantly increase neuron derivation from glial cells in vivo 83 .
Another risk factor to be considered in the context of in vivo conversion is the emergence of partially programmed cells, the potential tumorigenicity of such cells, and their potential impact on tissue homeostasis. Finally, as in vivo conversion efficiencies increase, depletion of the target cell population can become a serious issue. This is particularly true for astrocytes, oligodendrocytes, and microglia, which serve a plethora of vital functions in tissue homeostasis and neuronal function. In this context, cells with residual self-renewal capacity might serve as particularly attractive targets for neuronal conversion.

Implications
Notwithstanding the many fundamental and translational questions that remain to be addressed in the context of direct cell fate conversion, this field provides fascinating prospects for a number of biomedical applications ranging from disease modeling via drug discovery to cell therapy and endogenous regeneration ( Figure 1). For disease-related research, the prospect of age preservation in iNs could render these cells a preferred resource for patient-specific modeling of late-onset neurodegenerative disorders and establishing in vitro systems for compound screening. As for regeneration, somatic cell fate conversion and TF-based forward programming of PSCs could enable intricate approaches for generating neural subtypes faster and with much higher precision than conventional methods. Finally, in vivo transdifferentiation is about to revolutionize our concepts for neuroregeneration and, for some applications, might eventually substitute traditional cell transplantation strategies. However, although epigenetic remodeling is a general principle underlying cell programming, the preservation of somatic and age memory seems to be unique for each conversion paradigm. Thus, developing a better understanding of the mechanisms underlying specific conversion trajectories is essential in order to fully exploit this emerging technology for biomedical research and therapy.
In contrast to transplantation of in vitro-derived cells, in vivo cell fate conversion might enable restoration of neuronal circuitry from endogenous sources. NSC, neural stem cell; TF, transcription factor. 137.