Ever since antiquity the ideas of immortality, resistance to bodily injuries and the restoration of missing body parts have populated the myths of many civilizations. The ancient Greeks, for example, gave us the myth of Tithonus, immortal but still capable of aging; of Achilles who could not be injured – except at the proverbial heel – by either sword or spear; and Prometheus – bearer of fire – condemned by Zeus to regenerate his liver to perpetually feed voracious vultures. In America, Aztec records and even older Mayan mythology encompassed by the Popol Vuh are both abundantly imbued with the concept of perpetual regeneration (Bazzett, 2018). Irrespective of which civilization one may consider, it is highly improbable that such shared human beliefs were not inspired in some fashion by nature, which we know today has been a cauldron of biological invention for nearly 4 billion years. Thus, it is also extremely implausible that we have learned from Nature all that there is for us to know. On the contrary, we have but barely scratched the surface of biology. In fact, we do not know what is already possible. The sheer number of species out there waiting to show us what is indeed biologically possible is staggering. Equally remarkable is the fact that our species has the necessary tools to decode and understand them all if we so wished.
For the past 20 years, my laboratory has exploited the diversity of animal life to address the problem of regeneration. The central question for us is: why is regeneration so broadly but unevenly distributed in the animal kingdom? Likewise, in those animals with robust regenerative capacities: do they share common mechanisms to restore missing body parts or did unique mechanisms emerge independently in every species? Both are fundamental questions awaiting satisfactory mechanistic explanations. In an effort to carry out a rigorous molecular dissection of regeneration, we chose to study an organism with extraordinary regenerative capacities, the free-living, fresh-water flatworm planaria, Schmidtea mediterranea (Figure 1). We reasoned that if the wild type phenotypes were already extraordinary, i.e., regenerating a complete animal from a random body fragment, perturbing such biology should yield even more extraordinary phenotypes that would help illuminate the mechanisms underpinning regeneration.
RNA-mediated genetic interference (RNAi) provided us with a tool to perturb gene function (Sánchez Alvarado and Newmark, 1999) and regeneration screens were performed which identified hundreds of genes involved in regeneration (Reddien et al., 2005) and uncovered novel functions for key embryonic genes in an adult context (Arnold et al., 2019; Arnold et al., 2021; Gurley et al., 2008; Rink et al., 2009).
A key source of these animals’ regeneration prowess is a group of abundant, adult stem cells known as neoblasts. These cells are the only known cells to proliferate in adult asexual planarians and thus can be completely eliminated by exposing the animals to ionizing radiation (Bardeen and Baetjer, 1904). When the stem cells are thus abrogated, animals survive for 3-4 weeks on the virtue of their differentiated cells, but as these turnover, the animals eventually lose structural integrity and die. We devised methods to purify planarian stem cells, defined their expression profiles by bulk and single-cell RNA sequencing, and discovered a remarkable diversity of transcriptional states associated with these stem cells (Zeng et al., 2018). We also were able to prospectively isolate the pluripotent neoblasts from this cell population and demonstrated that when a single isolated cell was injected into animals devoid of stem cells – and thus destined to perish –such cells could rescue the viability of the stem cell-deficient animals and restore all the animal’s functions and properties, including their capacity to regenerate (Figure 2).
Intriguingly, when we studied these cells under three different biological contexts (tissue homeostasis, in vivo clonal expansion and regeneration), we were surprised to see that the same cell type displayed significantly different expression profiles depending on the context in which they were operating (Figure 3A). Hence, we have shown these cells to be remarkably dynamic, constantly occupying diverse states of continuous fate determination. Because pluripotent stem cells are generally assumed to primarily exist transiently in early embryogenesis, and can only be perpetuated artificially in vitro, our findings that pluripotent stem cells can be maintained in adult animals despite showing distinct transcriptional changes dictated by either physiological homeostasis and/or injury, are all the more provocative. This led us to propose a probabilistic model of stem cells to explain the plasticity of genomic output (transcriptome) displayed by these cells (Figure 3B). In this model, self-renewal becomes a conceptual property not permanently possessed by a discrete population, but transiently held by a small number of cells and arising probabilistically depending on the demands of the animal (Adler and Sánchez Alvarado, 2015). If these stem cells stochastically express progenitor markers for specific organs, perhaps injury induces changes in the frequency or periodicity of expression, resulting in altered differentiation of stem cell progeny. Such a model allows us to frame the remarkable plasticity of planarian in terms of dynamic cell states rather than statically-defined cell types.
We then wondered whether the ability of stem cells to occupy multiple transcriptional states would be a property shared by postmitotic differentiated cells. To address this problem, we generated a comprehensive atlas of whole-body regeneration in S. mediterranea (Benham-Pyle et al., 2021). This work revealed the existence of wound-induced cell states. An analysis of ~300,000 single-cell transcriptomes captured from regeneration-competent and regeneration-incompetent tissues identified transient regeneration-activated cell states (TRACS) in the muscle, epidermis and intestine. We also found that TRACS occurred only in post-mitotic cells and that their manifestation did not require cell division per se. TRACS were also characterized by distinct spatiotemporal distributions, and RNAi depletion of TRACS-enriched genes produced specific regeneration defects, depending on the tissues targeted. Muscle TRACS were found to be essential for tissue polarity, while epidermal TRACS were important for stem cell proliferation and endodermal (gut) TRACS were found to regulate stem cell proliferation and tissue remodeling (Figure 4). Our results uncovered that regenerative ability can emerge from coordinated transcriptional plasticity across adult derivatives of all three germ layers (Benham-Pyle et al., 2021).
Concluding Remarks
Our work on regeneration and in planarians has revealed previously unsuspected properties of both stem and postmitotic, differentiated cells. First, our work provides strong evidence that adult undifferentiated and differentiated cells possess unappreciated plasticity and can exist in multiple transcriptional states. In light of the current extensive reliance on induced pluripotent stem cells (iPSCs) to produce differentiated cells for therapeutic interventions in regenerative medicine it is appropriate to consider whether or not these reprogrammed cells possess the ability to occupy different transcriptional states.
Second, the extensive adoption and growing use of single cell RNA sequencing is providing a strong body of evidence for the existence of reproducible transcriptional states elicited by different biological conditions. As such, it is also appropriate to ask how homogeneous or not are the transcriptional profiles of differentiated iPSCs after their transplantation and integration into host tissues. Given that it should be possible to label iPSCs and thus follow their fates in host tissues, it would be interesting to purify these cells after they have been functioning in host tissues for a reasonable period of time and determine their single cell transcriptional profiles. Will differentiated iPSCs possess more, less, or no transcriptional plasticity? What would those profiles look like when the host tissues are challenged by conditions in which injury or disease are introduced? Understanding whether the iPSC postmitotic differentiated progeny behave distinguishably or indistinguishably from host differentiated cells should help us better understand whether unsuspected limitations of iPSCs may exist that may limit their long-term utility as therapeutic agents.
Third, our data provide strong evidence for us to question what is meant in adult organisms by “terminal differentiation”. Given the discovery of TRACS, that these transient transcriptional changes occur in postmitotic cells, and that they have essential roles in promoting regeneration, would it not be more accurate to think of “terminal differentiation” as “stable differentiation” instead? The implications here for regeneration biology and regenerative medicine are that if some cells are more stably differentiated than others, then their ability to regenerate or be restored may be more difficult. One corollary would be to ask whether differentiated iPSCs integrated in tissues are “ultra-stable” or possess limited transcriptional plasticity and therefore are less fit than wild type adult cells to respond to environmental insults, for example.
Finally, one important conclusion from our work is that revealing the mechanisms promoting and suppressing stable differentiation states will likely be essential to better understand the differentiated state of adult cells. This has two important implications. First, a clear mechanistic understanding of how stable differentiation is promoted and suppressed will shed light on why regenerative capacities are broadly but unevenly distributed across animals, including humans. And second, it may help us identify better ways to generate mature, differentiated, postmitotic iPSCs for regenerative medicine.
Acknowledgments:
I would like to thank all members of my laboratory, past and present, for their dedication, invaluable insights and scientific accomplishment in helping us advance our understanding of animal regeneration. I would also like to acknowledge Blair Benham-Pyle for providing Figure 4. ASA is an investigator of the Howard Hughes Medical Institute and the Stowers Institute for Medical Research.
References
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