Pluripotency retention and exogenous mRNA introduction in planarian stem cells in culture

Summary Planarians possess naturally occurring pluripotent adult somatic stem cells (neoblasts) required for homeostasis and whole-body regeneration. However, no reliable neoblast culture methods are currently available, hindering mechanistic studies of pluripotency and the development of transgenic tools. We report robust methods for neoblast culture and delivery of exogenous mRNAs. We identify optimal culture media for the short-term maintenance of neoblasts in vitro and show via transplantation that cultured stem cells retain pluripotency for two days. We developed a procedure that significantly improves neoblast yield and purity by modifying standard flow cytometry methods. These methods enable the introduction and expression of exogenous mRNAs in neoblasts, overcoming a key hurdle impeding the application of transgenics in planarians. The advances in cell culture reported here create new opportunities for mechanistic studies of planarian adult stem cell pluripotency, and provide a systematic framework to develop cell culture techniques in other emerging research organisms.


INTRODUCTION
While the control of pluripotency in animals has been examined in the germline and embryonic and induced pluripotent stem cells, no naturally occurring adult pluripotent stem cells have yet been identified in the roundworm, fly, fish, or rodent model systems. [1][2][3][4] By contrast, planarian flatworms, and acoels uniquely harbor an adult stem cell population, collectively termed neoblasts, 5 which includes a pluripotent subpopulation of clonogenic neoblasts 6-8 that enable whole-body regeneration and apparently limitless capacity for tissue homeostasis. The freshwater species Schmidtea mediterranea is a model for planarian development, regeneration, and, in particular, pluripotency in long-lived adult animals. 9 While regulators of pluripotency have been identified in neoblasts and studied using the RNA interference, 10-12 a lack of reliable culture methods and transgenesis limit in vivo exploration of adult stem cell pluripotency. 13 Indeed, published commentary has called for establishing reliable, standardized neoblast culture methods as an essential step in developing transgenic studies of planarians.
While genetic transformation strategies typically exploit early-stage embryos or cultured stem cells, [14][15][16][17][18][19][20] planarians instead reproduce asexually through neoblast proliferation and differentiation. 21 These cells can be transplanted into hosts lacking neoblasts (e.g., after lethal irradiation) to repopulate stem cells and rescue the host within one month of the irradiation. 6,8 Thus, transforming DNA or RNA into neoblasts before transplantation could produce transgenic planarians and subsequently enable significant breakthroughs in understanding the control of pluripotency in animals. However, despite numerous efforts, no reports currently describe the thriving culture of neoblasts or genetic modification of these animals. Based on the tremendous potential for planarian neoblasts to fill significant gaps in our understanding of regeneration in higher animals, we aimed to establish a robust culture method for pluripotent neoblasts that also enables efficient screening for successful delivery and transgenic expression of exogenous DNA or RNA.

RESULTS
KnockOut Dulbecco's Modified Eagle Medium with 5% CO 2 can maintain the pluripotency of neoblasts in vitro To establish standardized neoblast culture conditions, we first used an established back-gating method for flow cytometry sorting of X1(FS) cells, which typically contain approximately 23.4% G 2.5% neoblasts iScience Article (smedwi-1+) ( Figures 1A-1C). Testing both ambient atmosphere and 5% modified CO 2 conditions, we systematically screened 23 different types of media, including several commercially available mammalian and insect cell culture media, previously reported formulations (e.g., IPM and TTP), and dilutions of these media that better match osmolarity suitable for planarian cells (120 mOsm/kg) (Table S1). 22,23 To measure viability, cells cultured for one day were observed and stained with propidium iodide (PI), which labels the DNA of dead cells. We determined the percentage of PI negative cells by flow cytometry. Cells cultured in CMFB with or without 5% CO 2 modification displayed irregular cell surface morphologies accompanied by sizable cellular debris, suggesting poor viability ( Figure 1D). Consistent with microscopic evaluation, cells cultured in CMFB showed poor survival with or without 5% CO 2 modification (>60% dead cells) ( Figure S1A). In contrast, cells in all other conditions, such as IPM with or without 5% CO 2 , had normal morphology, suggesting high viability ( Figure 1D). Among all media conditions, seventeen formulations yielded viability higher than 60% ( Figure S1A). Notably, cells in Leibovitz's L-15 medium (L15) without 5% CO 2 extended long protrusions that were visible even after six days of culture ( Figure S1B), which suggested the occurrence of neuronal differentiation, as previously observed in cultured Caenorhabditis elegans embryonic cells. 24 To determine the proportion of neoblasts among total viable cells after 24 h of culture, we quantified the number of smedwi-1+ X1(FS) cells by fluorescent in situ hybridization (FISH). Notably, seven media with 5% modified CO 2 atmosphere maintained significantly more smedwi-1+ neoblasts than all other conditions, including diluted (d) Grace's medium, IPM, KnockOut DMEM, dL15 medium, dKnockOut DMEM, dSchneider's medium, and dDMEM ( Figure 1E). This result was supported by co-staining cells with smedwi-1 and the apoptotic/dead cell marker, Annexin V, which showed no detectable co-labeling, indicating that the neoblasts were viable ( Figure S1C). We next examined whether smedwi-1+ neoblasts persisted after three days in culture using these seven media +5% CO 2 , and observed that smedwi-1+ cells were present in all culture conditions tested here ( Figure S1D). Thus, these results showed that neoblasts could be maintained for at least three days in vitro. We, therefore, focused on testing dGrace's, IPM, KnockOut DMEM, dL15, dKnockOut DMEM, dSchneider's, and dDMEM media in subsequent optimization experiments.
Next, we assessed whether cultured neoblasts could divide in vitro. Although we did not observe any noticeable increase in cell number, low levels of both symmetric and asymmetric neoblast divisions were observed in cells cultured for one day, as determined by cell pair size and distribution of smedwi-1 transcripts ( Figure 1F). 12,25 Time-lapse microscopy imaging of X1(FS) cell behavior confirmed that neoblasts could divide in vitro. Both symmetric and asymmetric cell divisions were observed within the first 24 h in IPM, KnockOut DMEM, and dL15 medium, but not in the other four media tested (One in 300 X1(FS) cells) ( Figure 1G and Videos S1 and S2). PCNA + staining assays further suggested that the proliferating cells in IPM, KnockOut DMEM, and dL15 medium were significantly more than those in CMFB, Schneider's, and DMEM medium ( Figure S1E). These results suggested that a fraction of X1(FS), smedwi-1+ cells can  To determine if X1(FS) neoblasts can divide in vivo following in vitro culture, we transplanted X1(FS) cells cultured in the seven different media supplemented with 5% CO 2 for one, two, or three days. At eight days post-transplantation (dpt), the presence or absence of smedwi-1+ neoblast colonies and the number of smedwi-1+ neoblasts in each colony were determined. All X1(FS) neoblasts cultured for one or two days efficiently proliferated in vivo, except for those cultured in dGrace's medium with 5% CO 2 ( Figures 1H and  S2C). By comparing the number of smedwi-1+ neoblasts in each transplantation, we found that X1(FS) cells cultured for one day in either IPM or KnockOut DMEM formed the largest colonies in vivo ( Figure 1H). It was noticed that the cell number in transplanted cells that formed colonies was less than transplanted cell number. The efficiency of cell engraftment and viability after transplantation remains to be carefully determined in future studies of neoblast niches. In summary, cells grown in IPM and KnockOut DMEM performed best following one day of culture, but performed similarly to those grown in dKnockOut DMEM, dSchneider's, dL15, and dDMEM after two days of culture. In addition, the clonogenic capacity of X1(FS) neoblasts diminished considerably following three days in culture, regardless of the medium used. These results suggested that IPM, KnockOut DMEM, dL15, dKnockOut DMEM, dSchneider's, and dDMEM could all maintain neoblast proliferation potential for up to two days in culture in the presence of 5% CO 2 .
To evaluate the functional pluripotency of neoblasts cultured in these six media (IPM, KnockOut DMEM, dKnockOut DMEM, dL15, dSchneider's, and dDMEM), we assessed their ability to rescue lethally irradiated hosts following bulk-cell transplantation. Transplantation of non-cultured, freshly collected X1(FS) cells resulted in the rescue of 30-50% of the lethally irradiated (6,000 rad) sexual S. mediterranea hosts ( Figure 1I). X1(FS) cells cultured in IPM, dL15, or KnockOut DMEM for one or two days could also rescue hosts that were depleted of stem cells ( Figure 1I). Genotyping PCR and restriction fragment-length polymorphism (RFLP) assays were conducted to test whether sexual hosts had been transformed into the asexual biotype following transplantation with asexual neoblasts (Figures S2D-S2G). 8 Among the transplanted cultures, cells grown in KnockOut DMEM exhibited the highest and most robust host rescue ( Figure 1I). In summary, KnockOut DMEM with 5% CO 2 represented the most stable conditions for maintaining pluripotent neoblasts in culture for two days. Neoblasts grew in IPM and dL15 medium also retained their pluripotency for up to two days in culture, albeit with reduced rescue rates in irradiated host animals after transplantation.
SiRNeoblasts as an alternative source of transplantable neoblasts for primary culture To enrich neoblasts for culture, we tested three major types of cell-permeable DNA stains to enrich neoblasts in the G2/M cell cycle phases (DRAQ5, Vybrant DyeCycle, and SiR-DNA). The DNA stain, SiR-DNA, exhibited low cytotoxicity and resulted in 60% enrichment for smedwi-1+ neoblasts (Figures 2A, 2F, 2G, and S3A-S3C). 26 Comparison of smedwi-1+ and smedwi-1-cell morphology in the isolated populations showed that smedwi-1+ cells were generally larger than smedwi-1-cells ( Figure 2B). To discriminate between small and large cells in the SiR-DNA + population, the cytoplasmic dyes Cell Tracker Green (CT) and Calcein AM (CAM) were tested in combination with SiR-DNA for neoblast isolation ( Figures 2C and 2D). This dual dye staining strategy resulted in a significant increase in neoblast enrichment, indicated by the proportion of smedwi-1+ cells in FISH assays ( Figures 2E and 2F). In particular, SiR-DNA/CT co-staining showed comparable performance to Hoechst 33342 for enriching smedwi-1+ neoblasts ( Figure 2F), which we designated SiRNeoblasts, as previously described. 27 Unlike neoblasts obtained by Hoechst 33342 sorting, SiRNeoblasts proliferated and underwent colony expansion in vivo after transplantation into lethally irradiated planarians ( Figure 2G). Importantly, no noticeable differences in colony size were observed at 7 dpt among unstained X1(FS), single (SiR-DNA)-, or double (SiR-DNA/CT)-stained populations ( Figure 2G). Recently, a single-cell RNA sequencing study reported similarities between SiRNeoblasts and X1 cells, supporting that SiRNeoblasts could serve as an alternative cell source for functional studies of the neoblasts. 27 To characterize the proportions of SiRNeoblasts in different cell cycles, we stained these cells with Hoechst 33342. Since co-staining with Hoechst and SiR-DNA blocked the SiR-DNA signal, we instead used Hoechst staining in SiR-DNA-sorted neoblasts and found that 17.89% of SiRNeoblasts were in the G1, 13.02% at S, and 69.09% at G2/M cell cycle phases. (Figures S3D-S3G    iScience Article SiR-DNA staining facilitated the observation of the chromosomal separation dynamics of dividing SiRNeoblasts in vitro (Videos S3, S4, and S5), which confirmed the occurrence of bona fide cell division under the tested culture conditions. Both freshly isolated SiRNeoblasts and those cultured for one day in KnockOut DMEM with 5% CO2 could rescue lethally irradiated planarians at comparable rates to those reported for X1(FS) cells ( Figure 2H). We found that 27.2% of SiRNeoblasts express tgs-1, a gene expressed in planarian pluripotent stem cells and neural progenitors, 25,29,30 for as long as two days in KnockOut DMEM with 5% CO 2 ( Figure S4A). In addition, we observed no positive effects of co-culturing differentiated X1(FS) cells with SiRNeoblast ( Figure S4B). Based on these findings, we concluded that SiR-DNA/CT dual label-based cell sorting could be used to isolate clonogenic, pluripotent neoblasts. Moreover, these isolated SiRNeoblasts can be maintained in primary culture and serve as donor cells in transplantation assays.

Exogenous mRNA delivery by electroporation
Following the optimization of in vitro culture conditions to maintain neoblast pluripotency, we next tested different conditions for the delivery of exogenous (nucleic acid) molecules into neoblasts to attempt the genetic transformation of planarians. To this end, we first used dextran-FITC as a fluorescent indicator of membrane permeation to screen for the most suitable electroporation conditions of Hoechst 33342stained neoblasts ( Figure S5A). We tested 52 electroporation programs and 10 different buffers using X1 cells 31,32 and found that dextran-FITC was most effectively delivered into neoblasts using IPM buffer with electroporation at 100-120V (Table S2 and Figures S5B-S5D). Similarly, applying this electroporation method to X1(FS) cells rather than Hoechst 33342-sorted X1 cells showed that dextran-FITC + populations could only be detected following electroporation at 110V and 120V. However, less than 6% of dextran-FITC + X1(FS) cells were smedwi-1+ neoblasts, and virtually no smedwi-1+ cells could be detected after one day of culture in KnockOut DMEM with 5% CO 2 ( Figure S5E). Consistent with the drastic reduction in smedwi-1+ cell viability post-electroporation, none of the donor X1(FS) cell populations subjected to more than 100V formed colonies following transplantation into lethally irradiated donors ( Figure S5F). We hypothesized that this failure was likely due to the low proportion of smedwi-1+ neoblasts in total X1(FS) cells, which was further reduced after electroporation.
We then sought to identify the optimal electroporation conditions to retain the viability of SiRNeoblasts following the introduction of foreign genetic material ( Figure 3A). Consistent with previous experiments, electroporation at 110V-120V was required for dextran-TMR internalization into SiRNeoblasts ( Figures 3B and 3C). Different from that in X1(FS), smedwi-1+ cells were more abundant in the electroporated (110V and 120V) SiRNeoblasts compared to X1(FS) cells, and the electroporated SiRNeoblasts persisted for one day in culture ( Figure 3D). In addition, the electroporated SiRNeoblasts could form colonies and rescue lethally irradiated hosts upon transplantation ( Figures 3E and 3F). However, 120V electroporation resulted in SiRNeoblast rescue of relatively fewer irradiated hosts, suggesting that high voltages negatively impact SiRNeoblast viability.
To assess whether exogenous mRNA could be delivered into SiRNeoblasts by electroporation, tdTomato mRNA was added to the electroporation reaction along with Dextran-FITC. Dextran-FITC-positive SiRNeoblasts were sorted and cultured in KnockOut DMEM with 5% CO 2 . To confirm the successful delivery of mRNA, we probed cells via FISH at 20 h after electroporation and found detectable tdTomato mRNA signal in cells electroporated at either 110V or 120V ( Figure 3G). However, co-staining with smedwi-1+ revealed that not all tdTomato mRNA + cells retained neoblast identity in culture. The number of SiRNeoblasts positive for both tdTomato mRNA and smedwi-1 expression was significantly higher after 110V electroporation than after 120V, which was a similar response to electroporation to that observed in X1 and X1(FS) cells ( Figure 3H). To confirm the intracellular localization of the tdTomato mRNA, RNase A was used to treat cultured neoblasts at 20 h post-electroporation. The results showed that the number of tdTomato mRNA + cells after electroporation at 110V was comparable in the groups with and without RNase A treatment. Both groups contained significantly more tdTomato mRNA + cells than the matched control groups without electroporation (p < 0.01) (Figures S5G and S5H). These findings indicated that Adjusted p values were calculated by one-way ANOVA with the Tukey test. *, 0.01 < p < 0.05; **, 0.001 < p < 0.01; ***, p < 0.001; n.s., not significant. See also Figure S5 and Nanoluciferase mRNA delivered by TransIT can be successfully expressed in differentiating SiRNeoblasts Unfortunately, tdTomato expression was undetectable by either microscopy or antibody staining in cultured neoblasts. A recent study reported that Nanoluciferase (NanoLuc) mRNA could be expressed in somatic planarian cells through Viromer or TransIT transfection. 33 We, therefore, suspected that the high sensitivity and low autofluorescence background of the NanoLuc reporter could provide a tractable approach for visualizing neoblast transgene expression. To confirm that NanoLuc was indeed translated in cultured SiRNeoblasts, we transfected SiRNeoblasts with NanoLuc mRNA using the TransIT system (Figure 4A). Culture medium supplements (sodium pyruvate, vitamin, and amino acids, see Table S1) were also included in the modified KnockOut DMEM to determine whether they could enhance the mRNA expression. 33 We found that NanoLuc expression levels were higher in SiRNeoblasts cultured in modified KnockOut DMEM with 5% CO 2 modified atmosphere than in cells grown under the same conditions without supplements ( Figure 4B). To confirm that neoblasts could also be maintained in modified KnockOut DMEM with 5% CO 2 , SiRNeoblasts were stained and their proportion was compared to that of smedwi-1+ cells after one or three days of culture in modified KnockOut DMEM with 5% CO 2 , KnockOut DMEM with 5% CO 2 , and Iso-L15 under ambient conditions. The results showed that SiRNeoblasts could be maintained in modified Knockout DMEM as well as the Knockout DMEM, but not the Iso-L15 ( Figures 4C and 4D), which combined with earlier findings that higher CO 2 was required for consistently high SiRNeoblast activity ( Figure 1E), led us to use modified KnockOut DMEM with 5% CO 2 modification in subsequent experiments.
After delivering the NanoLuc mRNA into neoblasts by the two different methods (TransIT transfection and electroporation), we found that the NanoLuc signal was only detectable following TransIT transfection (Figure 4E). We next sought to compare the efficiency of mRNA delivery between TransIT and electroporation. Electroporation at 110V delivered NanoLuc mRNA into 11.22% of neoblasts, similar to that of tdTomato mRNA ( Figure S5I). In contrast, TransIT transfection delivered NanoLuc mRNA into 100% of cells tested (Figures 4F and S6A). Also, the percentage of NanoLuc mRNA + cells after RNase A treatment was indistinguishable from those without RNase A treatment (100%) ( Figure S6A). Furthermore, compared with the fluorescent intensity of NanoLuc mRNA with poly-A tail, the signal was obviously weaker within SiRNeoblasts after the transfection of NanoLuc mRNA without a poly-A tail ( Figure S6B). These results  iScience Article indicated that TransIT can indeed deliver NanoLuc mRNA into SiRNeoblasts with higher efficiency than electroporation.
To confirm this methodology with other mRNAs, we used TransIT to deliver mRNA encoding NanoLuc, smed-histone3.3-2xflag, mCherry, or NanoLuc-mCherry. Surprisingly, none of these proteins were detectable by Western blot (Figures S6C-S6F). In addition, immunofluorescence staining of transfected SiRNeoblasts could not provide a definitively positive signal because of autofluorescence or non-specific antibody binding in planarian cells ( Figures S6G-S6I), although the nuclear-localized staining signals were only captured in smed-histone3.3-2xflag mRNA transfected cells ( Figure S6I). Using chemiluminescence signal imaging, we observed that the ratio of NanoLuc + cells was much lower in live planarian cells (68/ 200k) than in 293T cells (100/121) ( Figure 4G). Since luciferase chemiluminescence assays have reportedly extremely high sensitivity for detecting signals from a small proportion of positive cells, this meager ratio of NanoLuc + cells may explain the success of the high sensitive chemiluminescence, but the absence of immunofluorescent signals. However, NanoLuc-mCherry transcripts exhibited a measurably lower signal likely due to a relatively longer coding sequence for reduced transfection efficiency, which suggested that the signal was indeed due to NanoLuc transcript expression ( Figure 4H).
We next checked whether NanoLuc + cells retained their smedwi-1+ phenotype to further investigate the potential low translation efficiency. The results clearly showed that all the NanoLuc + cells in our experiments were neither smedwi-1+ nor SMEDWI-1+ ( Figures 4I, 4J, S6J, and S7) (note: CTG + indicates whether cells were alive at the time of fixation). Since silencing mechanisms to suppress exogenous gene expression have long been suspected as a confounding factor in neoblast transformation experiments, this phenomenon strongly suggested the function of an unknown mechanism in silencing the expression of exogenous mRNA in neoblasts. We compared NanoLuc levels in SiRNeoblasts with that in bulk live cells to test this hypothesis. After confirming the temporal dynamics of NanoLuc expression by chemiluminescence (Figure 4K), we also compared the signal between live bulk cells from non-irradiated and lethally irradiated planarian populations. The results indicated that live bulk cells exhibited considerably higher NanoLuc signal than that in SiRNeoblasts, even after the depletion of neoblasts by lethal irradiation (Figures 4L,  4M, and S6K), which was consistent with the findings of Hall et al. 33 This result indicated that the NanoLuc signal was derived almost exclusively from somatic cells, and further suggested that planarian neoblasts harbored a means of preventing exogenous nucleic acids before differentiation.

DISCUSSION
The inability to genetically transform planarians has posed a long-standing obstacle to researching this otherwise highly versatile model for pluripotency and whole-body regeneration. The primary technical limitations underlying this obstacle involve determining the optimal culture conditions for maintaining pluripotent neoblasts and identifying an effective means for delivering exogenous nucleic acids into these cells. The cell culture system we have developed in this work resolves the former problem and enables further testing of strategies for exogenous material delivery, such as fluorescence-conjugated dextrans and mRNA, to ultimately demonstrate the translation of introduced mRNAs. Our method establishes the requisite foundation for developing transgenic and genome editing techniques in planarians to enable exciting new systematic investigations of naturally occurring pluripotent adult stem cell populations.
First, the use of SiRNeoblasts ensures the purity and viability of neoblasts, thus allowing relative ease in screening transgene delivery strategies. Moreover, we propose that positively charged polymers, not limited to TransIT, could be used to deliver larger molecules and genome-editing tools with higher transfection efficiency and a higher likelihood of obtaining transgenic animals.
Second, the low efficiency of transfection and translation may also be due to cultured cells' relatively decreased metabolic activity. The observed enhancement of NanoLuc mRNA translation following the addition of supplements suggested that the uptake and translation of mRNA depended on meeting metabolic requirements in cultured neoblasts. The cell culture platform described here provides a reliable approach for identifying nutrient requirements by comparing cultured neoblasts with in vivo neoblasts. Adding supplements to culture media can also optimize long-term culture systems and cell lines, enabling downstream research of transformation techniques and functional validation of other genetic manipulations (e.g., CRISPR RNPs for genome editing) in cultured cells. Meanwhile, the mechanism of why neoblast pluripotency maintenance requires 5% CO 2 has remained to be investigated. iScience Article Third, given that neoblasts are the de facto units of selection in planarians and that the viability of these animals heavily depends on their proper function and viability, it is logical that these cells have evolved robust molecular mechanisms to protect their genome from disruption by foreign nucleic acids. In the current study, we did observe that NanoLuc + cells were neither smedwi-1+ nor SMEDWI-1+, consistent with this hypothesis. Further experiments are necessary for definitive evidence supporting or refuting this hypothesis.
In summary, we describe a FACS isolation strategy and primary cell culture conditions for maintaining clonogenic, pluripotent neoblasts in vitro in short-term compatible with transplantation, repopulation, and rescue of lethally irradiated hosts. In addition, we demonstrate the successful introduction of exogenous mRNAs into neoblasts. Although further optimization is needed, this finding represents a significant technical milestone in developing protocols for generating transgenic planarians. Together with the findings of Hall et al., these results show that highly sensitive NanoLuc reporters can be robustly expressed in planarian cells. Interestingly, we found that cells labeled with traditional Hoechst 33342 staining, including X1, X2, and Xins, could not express NanoLuc, which supported the use of alternative means of obtaining neoblasts, such as SiRNeoblasts and CRNeoblasts, in further efforts to optimize neoblast culture conditions (Figure S6K). Our results also strongly suggest the presence of a long-suspected silencing mechanism in planarian neoblasts for suppressing exogenous gene expression, thus opening an avenue for further study into this potential mode of gene suppression and enabling the genetic transformation of regenerative planarian populations.

Limitations of the study
The cell culture condition we have developed in this study does not allow the long-term proliferation of neoblasts. Better cell culture conditions to solve this problem will be investigated in future studies. The current cell culture condition also did not show the detection of fluorescent protein, such as tdTomato and mCherry. The accomplishment of neoblast long-term culture may also provide a solution to increase the expression level of exogenously delivered genes and to achieve cell transformation for planarians.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:    Table S1 Calcium magnesium-free Medium, BSA N/A See Table S1 Isotonic Planarian Medium (IPM) N/A See Table S1 Teshirogi and Tohya Planarian Medium (TTP) N/A See

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Alejandro Sá nchez Alvarado (asa@stowers.org).

Materials availability
All reagents are available from the lead contact upon reasonable request. iScience Article 6,000 rads of g rays were used as transplant hosts. 8 After transplantation, hosts were maintained in Montjuïc water with 50 mg/mL of Gentamicin (GEMINI, 400-100P). For transplant rescue experiments, host animals were kept in 3.5 cm Petri dishes (1 worm/dish), and Montjuïc water was changed every 2-3 days.

Cell staining
Tails from planarians (>8 mm in length) were chopped into small pieces with a blade in Calcium Magnesium free buffer with 1% Bovine Serum Albumin (CMFB, Recipe in Table S1). The tissue pieces were then dissociated in CMFB for 20-30 min (no more than 30 min) with vigorous pipetting every 3-5 min until there were no visible tissue pieces. Dissociated cells were centrifuged at 290 3 g for 10 min at 4 C after being filtered through a 70 mm strainer. Cells were then resuspended in Isotonic Planarian Medium (IPM, Recipe in Table S1) with 10% Fetal Bovine Serum at 3 3 10 6 cell density for either Hoechst 33342 or SiR-DNA + CellTracker Green staining. To get X1 cells, dissociated cells in IPM (10% FBS) were stained with Hoechst 33342 (0.4 mg/mL, ThermoFisher Scientific, H3570) for 45 min. To enrich neoblasts, DRAQ5 (ThermoFisher Scientific, 62254, 5 mM), Vybrant DyeCycle Ruby stain (ThermoFisher Scientific, V10309, 10 mM), and SiR-DNA (1 mM, Cytoskeleton Inc., CY-SC007) were compared. To obtain SiRNeoblasts, dissociated cells were stained with SiR-DNA (1 mM, Cytoskeleton Inc., CY-SC007) for 1 h and then CellTracker Green CMFDA Dye (2.5 mg/mL, Thermo Fisher Technologies, C7025) for 10 min. SiR-DNA and CellTracker Green CMFDA Dye were resolved in DMSO. The final concentration of DMSO should be less than 0.1%. All staining was performed in the dark at room temperature. Tubes should be gently flicked every 5 min during the staining process to prevent cell aggregation leading to poor staining. Stained cells were centrifuged at 290 3 g for 10 min to remove dyes and resuspended with IPM (10% FBS). DAPI was added to the cell suspension at 1 mg/mL before loading on the cell sorter.

Flow cytometry and cell collection
The Influx or BD FACS Aria (FACAria Fusion SORP) cell sorter was set with a 100 mm tip/nozzle and multiple lasers (355, 488, 561, and 647 nm) in the collection mode. 0.4 X PBS was prepared as the sheath liquid by diluting 10 X PBS (Solarbio, P1022) with ultrapure water. The cells were sorted into 15 mL tubes at a flow rate of no more than 2. The number of sorted cells was set to meet the experimental requirements.

FACS of X1 and X1(FS)
X1(FS) cells were collected according to a pre-stained X1 population. 8,31 The gate in the BUV737 (355 nm, 740/35)/Hoechst 33342 (355 nm, 515/30) plot was set for the X1 population. To gate the X1(FS) cells, the X1 population from a control sample was used to define the forward scatter/side scatter gate.

FACS of SiRNeoblasts
SiRNeoblasts were collected after staining planarian cells with SiR-DNA and CellTracker Green CMFDA. 27 The gate in APC (647 nm, 670/30)/SSC plot was used to enrich the 4N cells. Populations with fluorescence intensity of FITC channel (488 nm, 530/30) within the top 60%-65% were selected as SiRNeoblasts. Sorted cells were centrifuged at 290 3 g for 10 min to remove the collection solution and resuspended with culture medium for further experiments.

Enrichment of bulk live cells
Dissociated cells without staining were filtered once through a 70-micron filter and twice through a 35-micron filter. Bulk cells in the flowthrough were centrifuged at 290 3 g for 10 min to remove the collection solution and resuspended with culture medium for further experiments.