Direct Cell Reprogramming and Phenotypic Conversion: An Analysis of Experimental Attempts to Transform Astrocytes into Neurons in Adult Animals

Abstract

Central nervous system (CNS) repair after injury or disease remains an unresolved problem in neurobiology research and an unmet medical need. Directly reprogramming or converting astrocytes to neurons (AtN) in adult animals has been investigated as a potential strategy to facilitate brain and spinal cord recovery and advance fundamental biology. Conceptually, AtN strategies rely on forced expression or repression of lineage-specific transcription factors to make endogenous astrocytes become "induced neurons" (iNs), presumably without re-entering any pluripotent or multipotent states. The AtN-derived cells have been reported to manifest certain neuronal functions in vivo. However, this approach has raised many new questions and alternative explanations regarding the biological features of the end products (e.g., iNs versus neuron-like cells, neural functional changes, etc.), developmental biology underpinnings, and neurobiological essentials. For this paper per se, we proposed to draw an unconventional distinction between direct cell conversion and direct cell reprogramming, relative to somatic nuclear transfer, based on the experimental methods utilized to initiate the transformation process, aiming to promote a more in-depth mechanistic exploration. Moreover, we have summarized the current tactics employed for AtN induction, comparisons between the bench endeavors concerning outcome tangibility, and discussion of the issues of published AtN protocols. Lastly, the urgency to clearly define/devise the theoretical frameworks, cell biological bases, and bench specifics to experimentally validate primary data of AtN studies was highlighted.

Keywords: astrocyte; direct cell conversion; direct cell reprogramming; functional multipotency of stem cells; neurodegeneration; neuron; neurotrauma.

Figure 1

Summary of the formulas used in this paper to describe direct cell conversion and direct cell reprogramming relative to the somatic nuclear transfer presentation. For this review, we have tried to distinctively describe direct cell conversion and direct cell reprogramming as per the experimental methods utilized to initiate the process (see text for rationales and purposes). In the current research field, however, the two terms are interchangeably used. (Left) Direct cell conversion is used here to describe the change of one somatic cell type (e.g., astrocyte) into another (e.g., iN) via the intervention of 1–2 transcription factors (TFs), presumably without passing through a multipotent or pluripotent-like state. It was proposed by some investigators as a straightforward method to attain the desired cell type from an already differentiated cell. (Middle) Direct cell reprogramming is defined by the manipulation of ≥3 TFs (or other factors including small molecules, signaling pathway modulators, etc.) that affect multiple epigenetic and/or genetic elements. (Right) Somatic nuclear transfer, the original concept of nuclear/cell reprogramming, is a process through which the nucleus of the somatic cell is removed and transplanted into an enucleated egg cell/ovum that subsequently becomes a zygote before being transplanted into a surrogate or gestational carrier. The new-born animal will be a DNA clone of the somatic cell nucleus donor. Note: the same set of scientific questions remain to be answered for either direct cell conversion or direct cell programming, despite the difference between their initiation recipes (see text for more discussions).

Figure 2

Schematic illustration of the general principles used to guide cell conversion or reprogramming for attaining neurons from astrocytes in adult animals. Common AtN conversion strategies were based on the manipulation of a single lineage-specific transcription factor (TF) to alter astrocytic chromatin accessibility and drive neuronal gene expression patterns in astrocytes. This was experimentally operated via two mechanisms: (1) the ectopic expression of a pioneer factor packaged into a viral vector; (2) the genetic knockdown of polypyrimidine tract binding protein 1 (PTBP1) using short-hairpin RNAs (shRNA), antisense oligonucleotides (ASO), or CRISPR-cas13d. Conversely, direct AtN reprogramming strategies utilize 3 or more TFs, small molecules, or signaling pathway modulators affecting multiple epigenetic and genetic elements of the starting astrocytes. For in vivo studies, both approaches relied on microinjection of the AtN induction factors/molecules into the target brain or spinal cord region. While the data and biological underpinnings of all AtN tactics remain to be fully validated/elucidated (indicated by question marks), it was suggested that direct conversion protocols induced a direct transition from one somatic cell type into another without passing through a multipotent or pluripotent state (orange dashed arrow) and direct reprogramming procedures were thought to return the starting cell to a more plastic and potentially multipotent-like state before triggering differentiation into the neuron-like phenotype (red dashed arrow; all images created using BioRender.com, accessed on: 17 November 2022).

Figure 3

Other therapeutic effects proposed to occur as a result of treatment with AtN protocols. (A) An overview of the post-injury microenvironment following TBI or SCI. The acute injury site consists of the activated astrocytes and microglia, infiltration of red blood cells (containing oxidants of Fe²⁺/Fe³⁺), white blood cells, and other immune cells, which jointly trigger oxidative stress/damage and neuroinflammation. These and other secondary injury events lead to reactive astrogliosis and formation of the glial scar, axonal degeneration, and breakdown of the blood–brain/spinal cord barrier (BBB/BSB). (B) AtN (astrocyte to neuron) induction has been reported to restore the BBB/BSB. (C) AtN protocols were shown to be capable of reversing the glial scar to neural tissue. (D,E) Direct AtN reprogramming and conversion regimens were reported to exert homeostatic effects of lessening the detrimental inflammatory response and microglial activation post-injury or disease. However, it remains unclear which principle or mechanism can be used to determine if the iNs will be beneficial for the existing or spared neural circuit. Note: real and potential issues and queries with these claims are presented as comments or questions in (A–E) and discussed in the respective review sections.