African pygmy mouse iPSCs as a model for in vitro embryogenesis, interspecies chimerism, and blastocyst complementation
Pjeter Gjonlleshaj, Ajda Lenardič, Monika Tarnowska-Sengül, David Taborsky, Christine L. Trautmann, João Agostinho de Sousa, Nicola Bundschuh, Frédéric Veyrunes, Ataman Sendoel, Ori Bar-Nur
TL;DR
Researchers created stem cells from African pygmy mice that can form chimeras with house mice, offering new insights into development and regenerative medicine.
Contribution
The study introduces chimera-competent iPSCs from African pygmy mice, enabling interspecies chimerism and blastocyst complementation.
Findings
APM-iPSCs differentiate into multiple cell types and form gastruloids in vitro.
Interspecies chimeras showed APM contributions to various tissues, including germ cells.
Blastocyst complementation produced APM muscle stem cells in mice.
Abstract
Somatic cell reprogramming into induced pluripotent stem cells (iPSCs) has been achieved in various mammals; however, assessing iPSC contribution to full-term chimeras beyond laboratory rodents remains challenging. Here, we demonstrate induction of pluripotency in male and female fibroblasts from the African pygmy mouse (APM), one of the smallest mammals. Using transcription factors and small molecules, we derived expandable APM-iPSCs that express pluripotency markers, differentiate into various cell types in vitro, and form gastruloids. Injection of APM-iPSCs into house mouse blastocysts generated full-term interspecies chimeras demonstrating extensive contribution to various tissues, including testicular germ cells. Notably, high APM contribution in organs such as heart and testes correlated with reduced organ size compared with mouse organs. Lastly, injection of APM-iPSCs into mouse…
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Taxonomy
TopicsPluripotent Stem Cells Research · Reproductive Biology and Fertility · CRISPR and Genetic Engineering
Introduction
The generation of animal chimeras, composed of two genotypes, has been a seminal milestone in developmental biology, advancing research in transgenesis and related fields.1^,^2^,^3 Chimeras are typically produced by embryo aggregation, introduction of inner cell mass (ICM) cells into embryos, or injection of pluripotent stem cells (PSCs) into early embryos.1^,^3 Intraspecies chimeras generally form more efficiently than interspecies chimeras, which often face mismatched developmental timing, increased cell competition, xenogeneic cell death, and immunological incompatibility across species.1^,^3^,^4 Despite these barriers, adult interspecies chimeras have been historically generated by embryo aggregation or cell transplantation between chicken and quail, sheep and goat, and house mouse (Mus musculus) and Ryukyu mouse (Mus caroli).5^,^6^,^7 Moreover, the generation of full-term interspecies chimeras using embryonic stem cells (ESCs) was achieved by injection of wood mouse (Apodemus sylvaticus) ESCs into house mouse blastocysts8 and between house mouse and brown rat (Rattus norvegicus), further demonstrating in these studies the utility of blastocyst complementation to generate xenogeneic organs in mouse-rat chimeras.9^,^10^,^11^,^12
These findings rekindled interest in interspecies chimerism as a potential method to generate human organs in animal hosts, leading to studies reporting on exclusive xenogeneic organ or cell generation using blastocyst complementation in an intraspecies or interspecies manner.13 Interestingly, although iPSC lines have been established from multiple species, most studies reported on PSC contribution to embryonic rather than full-term interspecies chimeras.1^,^13 Aside from reports on derivation of house mouse and rat interspecies chimeras, one notable study documented the contribution of Ryukyu spiny rat (Tokudaia osimensis) iPSCs to full-term interspecies chimerism with the house mouse.14 More recently, bat-iPSCs were reported to contribute to bat-chick postnatal chimeras15 and African pygmy mouse (APM)-iPSCs to full-term house mouse-APM chimeras.16 Several factors may preclude PSCs from contributing to interspecies chimerism,1 including culture conditions that play an important role in enabling PSCs to be chimera competent.17^,^18^,^19^,^20 In addition, constraints arise from technical limitations associated with the production of large animal chimeras and the restricted ability of certain primed or naive iPSC lines to extensively contribute to chimerism.1^,^3^,^13
Here, we set out to identify improved reprogramming conditions for the derivation of iPSCs from the APM, one of the smallest mammals, which exhibits a unique sex determination strategy. Specifically, female APM can harbor three different sex chromosome configurations: XX, XX∗, and X∗Y.21^,^22^,^23 It is hypothesized that an unknown factor on the X∗ chromosome mitigates the masculinizing effect of the Sry male determination gene on the Y chromosome, thereby enabling the development of X∗Y females.22^,^23 This represents an atypical strategy for sex determination, as mammals predominantly exhibit conserved sex specification, with any variation resulting in sterility. Given its small size, the APM also presents a unique model to assess variability in organ and animal size in interspecies chimeras. In particular, the potential to generate specific organs or cell types by blastocyst complementation in significantly larger mice or rats presents an attractive question. Collectively, uncovering conditions for the generation of full-term chimera-competent APM-iPSCs may help elucidate questions in developmental biology related to cross-species barriers in interspecies chimeras, size regulation, and sex determination.
Results
Small-molecule screen unveils enhanced conditions for the derivation of APM-iPSCs
We initiated our study by setting out to identify conditions for reprogramming APM fibroblasts to pluripotency (Figures 1A and 1B). For this, we utilized lentiviruses expressing a doxycycline (dox)-inducible polycistronic cassette carrying the rat Oct4, Klf4, Sox2, and c-Myc genes (“LV-tetO-rOKSM”), in concert with constitutive reverse tetracycline-controlled transactivator 3 (rtTA3) (“LV-EF1α-rtTA3”) (Figures 1B and S1A). We confirmed successful transduction in male (XY) APM fibroblasts through OCT4 protein expression at 48 h after dox administration (Figures S1B and S1C). Additionally, we hypothesized that specific small-molecule cocktails could enhance the reprogramming efficiency of APM fibroblasts as they do in house mouse cells. For instance, ascorbic acid (A), the GSK-3 inhibitor CHIR99021 (C), with or without the TGF-β receptor inhibitor RepSox (R) (abbreviated as AC and ACR, respectively), substantially enhance reprogramming of mouse fibroblasts into iPSCs.24^,^25^,^26^,^27^,^28^,^29 Furthermore, dual inhibition of the MEK and GSK-3 pathways, commonly known as “2i,” enabled the capture of ground-state pluripotency and promoted the derivation of PSCs from species other than the house mouse including rats.28^,^30^,^31 Finally, a medium containing leukemia inhibitory factor (LIF), CHIR99021, dimethindene maleate, and minocycline hydrochloride (“LCDM”) gave rise to extended pluripotent stem cells (EPSCs) carrying enhanced ability to contribute to chimerism.32 In light of these studies, we set out to test whether these small-molecule cocktails can promote the reprogramming of APM fibroblasts into iPSCs (Figure 1B). To this end, transduced APM fibroblasts were seeded in either serum-containing ESC medium or serum-free N2B27 medium, both supplemented with LIF, various combinations of small molecules, and dox (Figure 1B). Several days after the initiation of reprogramming, we observed morphological changes indicative of a mesenchymal-to-epithelial transition (Figure S1D). Notably, by day 9 of reprogramming, we observed the formation of iPSC-like colonies under several conditions, and more prominently with AC, ACR, and LCDM (Figure 1C). These APM-iPSC-like colonies were alkaline phosphatase (AP) positive, an early marker for pluripotency,33 and showed increased AP staining with small-molecule treatment, particularly AC (Figure 1C). Concomitantly, we attempted the reprogramming of female (XX∗) APM fibroblasts and similarly observed that small molecules enhanced reprogramming as assessed by AP activity (Figure 1D).Figure 1. Generation and characterization of APM-iPSCs(A) Representative photo of an adult African pygmy mouse (Mus minutoides, APM).(B) Schematic of the experimental plan to unveil improved culture conditions for the derivation and maintenance of APM-iPSCs by screening small-molecule combinations together with transcription factor overexpression.(C) Representative phase-contrast (top) and alkaline phosphatase (bottom) images of male APM fibroblasts subjected to the indicated conditions at day 9. Scale bars, 500 μm.(D) Representative bright-field (top) and alkaline phosphatase (bottom) images of female APM fibroblasts subjected to the indicated conditions at day 4. Scale bars, 100 μm.(E) Representative phase-contrast images of APM-iPSC lines derived and maintained under the indicated conditions at passage 1. Scale bars, 100 μm.(F) Representative immunofluorescence images of OCT4, SOX2, and SSEA1 in male APM-iPSCs. Note that not all iPSC colonies express SSEA1. Scale bars, 100 μm.(G) Karyogram of male APM-iPSCs showing a normal number of chromosomes (2n = 18, 17/20 examined cells), indicative of the APM Stellenbosch clade.34^,^35(H) Representative immunofluorescence images of NANOG in female APM-iPSCs. Note that not all iPSC colonies express NANOG. Scale bars, 100 μm.
Subsequently, we tested whether dox withdrawal together with various small molecules would enable APM-iPSC-like cells to expand in an undifferentiated state. These culture conditions included ESC medium (“Serum+LIF”) with or without 2i, and N2B27 medium+LIF+2i, with or without a B-Raf inhibitor (Figure 1B).14^,^28 Using this approach, we successfully expanded APM-iPSC-like cells in ESC or N2B27 media, either using 2i or 2i+B-Raf inhibitor, resulting in the establishment of dome-shaped colonies resembling mouse iPSCs (Figures 1E and S1E–S1I). Of note, removal of 2i from the ESC medium led to morphological changes and differentiation of colonies, regardless of whether these APM-iPSC-like cells were cultured on irradiated mouse or APM fibroblast “feeder” layers (Figures S1G–S1I). The APM-iPSC-like colonies expressed the pluripotency markers OCT4, SOX2, and partially the surface marker SSEA1 (Figure 1F). Importantly, we verified that male APM-iPSC-like cells retained a normal diploid karyotype composed of 18 chromosomes, typically indicative of the Stellenbosch APM clade found in a distinct region in South Africa (Figure 1G).34^,^35 We also successfully expanded female (XX∗) APM-iPSC-like colonies in the Serum+LIF+2i condition, that expressed the pluripotency markers NANOG, OCT4, SOX2, and SSEA1, albeit not uniformly (Figures 1H and S1J–S1L). Lastly, we attempted iPSC generation using a non-integrative episomal plasmid encoding pluripotency factors36 in the presence of ACR, resulting in the establishment of female APM-iPSC-like cells that expressed OCT4 (Figures S1M–S1O). In summary, we developed improved conditions for the reprogramming of male and female APM fibroblasts into pluripotency, identifying molecules that supported their expansion in an undifferentiated state, leading us to conclude that APM-iPSCs were generated.
APM-iPSCs differentiate in vitro into various cell types and form gastruloids
Our results so far revealed that APM-iPSCs express pluripotency markers and can be expanded in an undifferentiated state; however, their capacity to give rise to specific differentiated cell types in vitro remained unexplored. To investigate this, we subjected APM-iPSCs to directed differentiation protocols that can give rise to either neuronal or cardiac cells through the formation of embryoid bodies (EBs) (Figure 2A).37^,^38 Utilizing non-adherent microwell plates, APM-iPSCs readily gave rise to spheroid EBs within a few days (Figure 2B). Subsequent exposure of the APM-EBs to the two differentiation protocols for several days, followed by re-plating onto adherent gelatin-coated plates, resulted in the generation of neuronal cells expressing NESTIN, or beating cardiomyocytes expressing the cardiac marker TROPONIN (Figures 2C–2E and Video S1). These results highlight the ability of APM-iPSCs to differentiate into two important cell types.Figure 2APM-iPSCs differentiate in vitro into multiple cell types and form gastruloids(A) Experimental overview of APM embryoid body (EB) formation and subsequent differentiation into neural cells and cardiomyocytes.(B) Representative phase-contrast image of an APM-EB in a microwell plate at day 2. Scale bar, 100 μm.(C) Representative phase-contrast image of APM-EBs plated onto gelatin-coated dishes. Scale bar, 100 μm.(D) Representative phase-contrast and immunofluorescence images of NESTIN^+^ neural cells derived from plated APM-EBs. Scale bars, 100 μm.(E) Representative phase-contrast and immunofluorescence images of cardiac TROPONIN^+^ cells derived from plated APM-EBs. Scale bars, 100 μm.(F) Experimental schematic showing the steps taken to generate mouse and APM gastruloids.(G) Representative phase-contrast images of mouse and APM gastruloids at the indicated time points. Scale bars, 100 μm.(H) Representative immunofluorescence images of T/BRACHYURY (T/BRA) in APM gastruloids at the indicated time points. Scale bars, 100 μm.See also Video S1.
Video S1. Contractile APM-iPSC-derived cardiomyocytes at day 10 of differentiation, related to Figure 2
Next, we wished to assess whether APM-iPSCs can give rise to gastruloids, in vitro-derived 3D structures that mimic early developmental stages, including gastrulation and symmetry breaking, which are characteristic of developing embryos.39^,^40 To this end, APM-iPSCs were suspended in low-adherent cell culture plates and subjected to conditions that favor gastruloid formation as described previously (Figure 2F).39 As a positive control, we subjected mouse ESCs to identical differentiation conditions. Within 72–96 h, we observed structure elongation, as expected during gastruloid formation, in both APM-iPSCs and mouse ESCs (Figure 2G). Notably, the elongation occurred faster with APM-iPSCs, as early as 72 h, and dissociated by 120 h (Figure 2G). Subjecting the mouse and APM gastruloid-like-structures to immunostaining for the gastrulation markers T (BRACHYURY) and SOX2 revealed a unique expression pattern indicative of symmetry breaking and gastruloid formation (Figures 2H and S2A). However, unlike mouse gastruloids, APM gastruloids lost this polarity by 120 h, potentially reflecting faster embryonic development in vitro (Figures 2H and S2A). In conclusion, our findings demonstrate that APM-iPSCs harbor a capacity to differentiate into ectodermal and mesodermal cell types in vitro through EBs and are capable of forming gastruloids with anterior-posterior polarity and symmetry breaking. These results underscore their potential as a model for studying early APM embryonic development in vitro.
Investigating the in vivo differentiation potential of APM-iPSCs in interspecies chimeras
The in vivo contribution of PSCs to various cell types and organs in chimeras is a key assay to evaluate pluripotency.1 Since APM transgenic procedures including PSC injections into APM blastocysts do not exist, we wished to capitalize on existing transgenic procedures in the house mouse to assess whether APM-iPSCs could contribute to in vivo embryonic or postnatal development in house mouse-APM chimeras (mouse-APM chimeras) (Figure 3A). To enable the identification of APM-iPSC contribution to various internal organs in vivo, we pre-labeled APM-iPSCs with lentiviruses encoding H2B-red fluorescent protein (H2B-RFP) under a constitutive EF1α promoter (LV-EF1α-H2B-RFP) (Figures 3A–3C). This labeling system previously enabled us to detect RFP positive (RFP^+^) mouse or rat PSC-derived cells in embryonic or adult chimeras.41 For this trial, we selected APM-iPSCs that were generated using the LCDM condition, as it enables PSC contribution to chimerism even from single cells.32 Given the LCDM condition contains minocycline (M), which can activate the tetracycline-response element of the dox-inducible LV-tetO-rOKSM cassette, we opted to culture LCDM-derived APM-iPSCs in Serum+LIF+2i and proceeded to inject fluorescence-activated cell sorting (FACS)-purified RFP^+^ APM-iPSCs into albino mouse blastocysts to generate chimeras (Figures 3A–3C).Figure 3APM-iPSC contribution to interspecies chimerism with the house mouse(A) Schematic of experimental design to generate embryonic or adult mouse-APM interspecies chimeras.(B) Flow cytometry analysis of H2B-RFP^+^ APM-iPSCs.(C) Representative phase-contrast and RFP overlay images of FACS-purified H2B-RFP^+^ APM-iPSCs. Scale bars, 100 μm.(D) Representative bright-field and H2B-RFP overlay images of high- and low-grade interspecies E11.5 mouse-APM embryonic chimeras, as well as three non-chimeric embryos. Chimerism was determined by RFP expression. Scale bars, 5 mm.(E) Representative photos of a full-term mouse-APM chimera and a non-chimeric mouse littermate at the indicated ages.(F) PCR analysis using mouse- or APM-specific primers for the Gh gene in the indicated animals.(G) Sanger sequencing and alignment of the PCR product sequences shown in (F).(H) Representative bright-field and RFP overlay images of the indicated organs harvested from an 11-week-old mouse-APM adult chimera. Only a representative portion of the liver is shown. Look-up tables (LUTs) were adjusted individually for each image. Scale bars, 5 mm.(I) Graphical illustration presenting the effort to establish a secondary iPSC reprogrammable system from H2B-RFP^+^ dermal cells isolated from an interspecies chimera.(J) Representative phase-contrast and RFP overlay images of dermal cells isolated from mouse-APM chimera skin. Note the nuclear H2B-RFP expression in APM dermal cells. Scale bars, 100 μm.(K) Representative phase-contrast and RFP overlay images of reprogrammable APM dermal cells subjected to the indicated reprogramming conditions and imaged at day 11. Scale bars, 500 μm.(L) Representative immunostaining images of OCT4 in dox-independent 2^nd^ system-derived APM-iPSCs. Scale bars, 100 μm.(M) Representative karyogram showing a normal APM karyotype (2n = 18 chromosomes), obtained from reprogrammable RFP^+^ dermal cells derived from a mouse-APM chimera.
To assess the contribution to embryonic chimerism, we analyzed fetuses at E11.5, detecting H2B-RFP expression in two embryos (Figures 3D and S3A). Concurrently, we enabled female mice carrying embryos to give birth and monitored the newborn pups for signs of chimerism based on fur color. Among one of the litters, we identified a pup that had both white and dark fur, indicating successful contribution of APM-iPSCs to the development of a full-term interspecies mouse-APM chimera (Figure 3E). The chimera appeared healthy and was surprisingly comparable in size to mouse littermates, despite the chimeric contribution of APM-iPSCs (Figure 3E). We further confirmed that it was an interspecies chimera through PCR genotyping and Sanger sequencing for mouse- and APM-specific alleles of the growth hormone (Gh) gene (Figures 3F and 3G). At 11 weeks of age, we detected extensive APM-iPSC contribution to numerous organs using the RFP reporter, including the brain, heart, lung, kidney, liver, and cauda (Figures 3H and S3B).
As the next step, we wished to determine whether a reprogrammable APM cell line can be established from the mouse-APM chimera. This cell line is expected to harbor the dox-inducible lentiviral vector cassette and express reprogramming factors upon dox exposure, enabling the derivation of secondary APM-iPSCs (Figure 3I). For this, we extracted dermal skin cells from the mouse-APM chimera to establish RFP^+^ dermal-derived cells in vitro (Figures 3J and S3C). We then FACS-purified RFP^+^ dermal skin cells and obtained a cell line predominantly composed of chimera-derived RFP^+^ APM dermal cells (Figure S3D). Subjecting the RFP^+^ APM dermal cells to dox and small molecules gave rise to RFP^+^ iPSC colonies, demonstrating the establishment of a secondary reprogramming system, as previously reported for mouse and human cells (Figure 3K).42^,^43^,^44 However, we also documented the emergence of RFP^−^ or mixed RFP^+^/RFP^−^ APM-iPSCs, suggesting silencing of the lentiviral vectors as previously observed (Figure 3K).41 Notably, the dox-independent iPSCs expressed OCT4 and maintained RFP expression (Figure 3L). Finally, RFP^+^ dermal cells showed a normal karyotype, albeit we also surprisingly observed cells with a tetraploid karyotype, requiring further investigation (Figures 3M and S3E). In conclusion, APM-iPSCs can form interspecies chimeras with the house mouse, and their contribution was detected in several organs. Furthermore, we established a secondary APM reprogrammable system, representing to our knowledge, the first establishment of such a system through interspecies chimerism.
Dissection of APM-iPSC contribution to organ size and germ line in chimeras
The observation that lentiviruses are silenced in APM-iPSCs prompted us to engineer a different reporter system to detect APM-iPSC contribution to interspecies chimerism. Capitalizing on the availability of the APM genomic assembly sequence (see STAR Methods), we employed CRISPR-Cas9 to genetically target the Rosa26 locus with H2B-mCherry (R26-H2B-mCherry) in male APM-iPSCs (Figure 4A).45^,^46^,^47 Following electroporation, a few single-cell-derived APM-iPSC clones expressed mCHERRY and were picked and propagated, or alternatively FACS purified to derive iPSC clones (Figures 4B, S4A, and S4B). Heterozygous or homozygous targeting of the Rosa26 locus was confirmed through PCR genotyping (Figure S4C). Additionally, we confirmed targeting of the construct into the APM Rosa26 locus using specific primers followed by sequencing (Figures S4D–S4G).Figure 4. Analysis of APM-iPSC contribution to organ size and the germline in chimeras(A) Genome engineering strategy to introduce an H2B-mCherry transgene into the Rosa26 locus of APM-iPSCs.(B) Representative phase-contrast and fluorescence overlay images of FACS-purified H2B-mCHERRY^+^APM-iPSCs. Scale bars, 100 μm.(C) Schematic of the blastocyst injection strategy used to generate interspecies mouse-APM chimeras.(D) Representative photos of deceased 10-week-old sex-matched mouse-APM chimeras. Scale bars, 1 cm.(E) Graph illustrating body weight of sex-matched or unmatched 10-week-old mouse-APM chimeras in comparison to control mice of the same age. Weight is presented as mean ± SD. Statistical analysis was performed for males and females separately using an unpaired two-tailed t test. ∗p ≤ 0.05.(F) Representative bright-field and fluorescence overlay images of the specified organs harvested from the indicated 10-week-old mouse-APM chimeras and control mice. Note that chimeric organs are shown on the right in each image (bottom for the spleen) and mouse control is shown on the left. Note also that control mouse organs are shown more than once, particularly for chimeras #1–3, as not all chimeras were analyzed on the same day. Additionally, a chimera liver is shown without a mouse control, which is provided in Figure S5. LUTs were adjusted individually for each image. Scale bars, 5 mm.(G) Graphs showing the weight of the indicated organs harvested from age- and sex-matched male mouse-APM chimeras vs. male control mice. The organ weight corresponds to the organs shown in (F) and is presented as mean ± SD. ∗p ≤ 0.05; ∗∗∗∗p ≤ 0.0001.(H) Representative immunostaining images of the indicated markers in cross-sections of mouse-APM chimera testes. Note that VASA- and PNA-expressing cells are of APM origin, as they co-express the H2B-mCherry transgene. Scale bars, 100 μm.See also Video S2.
Next, several established APM-iPSC clones were injected into B6 albino blastocysts, aiming to generate full-term interspecies chimeras (Figure 4C; Table S1). Judging by fur color and reproductive organs, this effort resulted in the birth of 4 XY-mouse/XY-APM male chimeras and 4 XX-mouse/XY-APM female chimeras out of a total of 42 born mice (19% efficiency) (Figures 4D and S5A; Table S1). These mouse-APM chimeras developed normally, with no detectable developmental defects or other abnormalities at week 10 (Figures 4D and S5A). We monitored the weight of the interspecies chimeras on a weekly basis for 7 weeks and observed no significant weight difference in comparison to control non-chimeric male or female littermates, aside from at 2 weeks of age, when non-chimeric mice weighed slightly more (Figure S5B). At week 10 of age, the XY-mouse/XY-APM chimeras were sacrificed and weighed, revealing a slight trend (p value = 0.063) toward reduced body weight in comparison to control male littermates (Figure 4E). We harvested various organs from the chimeras and investigated chimeric contribution based on the R26-H2B-mCherry reporter expression (Figures 4F and S5C). Interestingly, in the 4 XY-mouse/XY-APM chimeras, we documented increased APM-iPSC contribution to the brain, heart, and testes in comparison to the kidneys, liver, spleen, and lungs (Figure 4F and Video S2). Furthermore, the two organs that exhibited reduced weight in comparison to control native mouse organs were the heart and testes, which exhibited substantial chimerism based on R26-H2B-mCherry expression (Figures 4F and 4G). Remarkably, the testes contained VASA^+^/mCHERRY^+^ and PNA^+^/mCHERRY^+^ cells, demonstrating the contribution of APM-iPSCs to the male germ line in the testes of chimeras (Figure 4H). Moreover, the PNA^+^/mCHERRY^+^ cells exhibited a sperm head-like structure in developing spermatids (Figure 4H).
Video S2. Ex vivo contraction of the heart tissue from an XY-mouse/XY-APM chimera #4, related to Figure 4. Residual contractions of the explanted heart tissue are shown at increased zoom. Nuclear mCHERRY fluorescence signal is visible. The “14x” label is an acquisition software overlay and is not representative of the imaging magnification.
We proceeded to analyze the 4 XX-mouse/XY-APM female chimeras, which appeared normal and exhibited a similar body weight to non-chimeric female mouse littermates at week 10 (Figure 4E). Of note, we observed less chimerism based on the R26-H2B-mCherry reporter expression in internal organs of these chimeras in comparison to the sex-matched chimeras, with detectable chimeric contribution mostly confined to the heart and brain (Figure S5C). However, unlike sex-matched chimeras, these chimeras did not exhibit reduced weight in chimeric organs, most likely due to the limited chimeric contribution (Figure S5D).
Next, we established an mCHERRY^+^ APM dermal cell line from a chimera (Figures S6A–S6D). Upon exposure to dox and small molecules, dermal mCHERRY^+^ cells reprogrammed into iPSC-like colonies, which were then picked and expanded in the absence of dox while maintaining OCT4 expression, confirming the establishment of a secondary reprogramming system (Figures S6E–S6G). Finally, we wished to determine whether female APM-iPSCs could contribute to interspecies chimerism with the house mouse. To investigate this, we targeted the Rosa26 locus in XX∗ APM-iPSCs with an H2B-EGFP vector using CRISPR-Cas9, successfully deriving two clones carrying either a homozygous or heterozygous transgene integration (Figures S6H and S6I). We proceeded to inject these clones into B6 albino blastocysts, an effort that resulted in the generation of a single low-grade interspecies female chimera based on dark fur (Figures S6J and S6K; Table S1). In conclusion, we successfully edited the APM genome using CRISPR-Cas9, generating both male and female APM-iPSCs carrying transgenic reporters. We centered our attention on male APM-iPSCs, which contributed extensively to interspecies chimerism, including the formation of testicular germ cells. Moreover, we observed a correlation between the extent of APM-iPSC contribution to specific organs and reduced organ weight. We postulate that APM cells contributed to this phenotype, given the inherently small size of APM relative to the house mouse.
Ablation of mouse PAX7+ cells facilitates APM satellite cell generation in chimeras
Blastocyst complementation represents an attractive approach to exclusively produce desired organs, tissues, or cell types from PSCs in intra- or inter-species chimeras.13 This method typically relies on genetic mutations in host embryos or ablation of host-derived cells, resulting in an “empty” developmental niche that can be colonized with PSC-derived cells.13 As the APM is evolutionarily closer to the house mouse than the rat, we wondered whether APM-iPSCs can exclusively produce a xenogeneic APM cell type in interspecies chimeras via blastocyst complementation. To this end, we utilized house mouse blastocysts carrying a Pax7-Cre/ERT2; Rosa26-loxP-STOP-loxP-Diphtheria toxin A (Pax7^Cre/ERT2^; Rosa26^LSL-DTA^) ablation system, designed to eliminate PAX7-expressing cells by tamoxifen.48 As PAX7 is uniquely expressed in muscle stem cells termed satellite cells,49 its ablation in chimeras during postnatal development is expected to enable substantial colonization of the niche with PSC-derived satellite cells.50 We previously employed this genetic ablation system to demonstrate exclusive generation of mouse iPSC-derived satellite cells in intraspecies chimeras.50 Building upon this, we proceeded to inject R26-H2B-mCherry APM-iPSCs into Pax7^Cre/ERT2^; Rosa26^LSL-DTA^ mouse blastocysts of a B6 genetic background, which give rise to mice with dark fur (Figure 5A). As a control, we analyzed B6 albino mouse-APM chimeras (presented in Figure 4) lacking the transgenic cell ablation system (Figure 5A).Figure 5. Ablation of mouse PAX7^+^ cells promotes APM satellite cell formation in mouse-APM chimeras(A) Schematic of experimental design to preferentially obtain APM satellite cells in mouse-APM chimeras through elimination of host mouse PAX7^+^ cells during postnatal growth.(B) Representative photos of mouse-APM chimeras generated from Pax7^Cre/ERT2^; Rosa26^LSL-DTA^ blastocysts. Scale bars, 1 cm.(C) Representative bright-field and fluorescence overlay images of tibialis anterior (TA) muscles from the indicated animals. Scale bars, 5 mm.(D) Immunostaining images of TA muscle cross-sections for the indicated markers in the specified chimeras. Note the presence of PAX7^+^/mCHERRY^+^ cells with and without tamoxifen treatment. Scale bars, 100 μm.(E) Representative FACS plots displaying the percentage of mCHERRY^+^ satellite cells within the ITGA7^+^ satellite cell population of a tamoxifen-treated vs. a B6 chimera.(F) Graph showing quantification of the representative FACS plots shown in (E) for a larger group of chimeras. Data are shown as mean ± SD. N = 3 tamoxifen-treated chimeras and N = 8 B6 chimeras. ∗∗p ≤ 0.01.(G) Uniform manifold approximation and projection (UMAP) based on scRNA-seq of all cell populations in tamoxifen-treated mouse-APM chimera skeletal muscles, colored by the indicated cell type. SCs, satellite cells; FAPs, fibro-adipogenic progenitors.(H) Dot plot showing individual gene expression in mouse-APM cell populations used to annotate the UMAP shown in (G).(I) UMAP of all cells from skeletal muscles of a mouse-APM chimera, colored by detection of the H2B-mCherry transcript.(J) Representative bright-field and fluorescence overlay images of FACS-purified ITGA7^+^/mCHERRY^+^ and ITGA7^+^/mCHERRY^−^ myoblasts. Scale bars, 100 μm.(K) Representative immunostaining images for the skeletal muscle differentiation marker ACTN2 in ITGA7^+^/mCHERRY^+^ myoblast-derived myotubes. Scale bars, 100 μm.
This effort resulted in the generation of 3 Pax7^Cre/ERT2^; Rosa26^LSL-DTA^ mouse-APM chimeras, identified through brown fur patches in the back and white fur in the lower torso, a distinct characteristic of the APM abdomen, as Pax7^Cre/ERT2^; Rosa26^LSL-DTA^ B6 mice are entirely black (Figures 5B and S7A; Table S1). We injected these chimeras postnatally with tamoxifen from day 3 to 5 and then once per week for a total duration of 10 weeks. At this time point, the chimeras were sacrificed, and their skeletal muscles were analyzed, revealing nuclear H2B-mCHERRY^+^ cells in the tibialis anterior (TA) muscles of several analyzed chimeras (Figure 5C). Next, we sectioned the TA muscles and assessed the presence of APM-iPSC-derived PAX7^+^/mCHERRY^+^ cells in B6 albino vs. tamoxifen-injected chimeras. We readily detected PAX7^+^/mCHERRY^+^ APM satellite cells in both B6 albino and tamoxifen-injected chimeras (Figure 5D). To quantitatively assess whether ablation of mouse host PAX7^+^ cells facilitated the contribution of APM-iPSCs to the satellite cell niche, we used FACS to count the number of cells that expressed the satellite cell surface marker Alpha 7 integrin (ITGA7)51 and the R26-H2B-mCherry reporter, out of the total ITGA7^+^ satellite cells (Figures S7B and S7C). This analysis revealed that tamoxifen-treated chimeras exhibited a significantly higher number of ITGA7^+^/mCHERRY^+^ satellite cells (ca. 45%–75%) compared to approximately 15% mCHERRY^+^/ITGA7^+^ satellite cells in B6 albino chimeras (Figures 5E and 5F). We postulate that not all ITGA7^+^ satellite cells expressed mCHERRY due to transgene silencing, low transcription in quiescent cells, or incomplete elimination of mouse satellite cells by tamoxifen.
We subsequently set out to dissect the contribution of APM-iPSCs to the repertoire of resident cell populations in skeletal muscles of a tamoxifen-injected chimera through single-cell RNA sequencing (scRNA-seq). Based on established markers, we could identify endothelial cells, satellite cells, fibroblasts, immune cells, and other cell types characteristic of skeletal muscle tissue (Figures 5G and 5H). As sequence similarity between mouse and APM cells rendered it challenging to distinguish between the two species, we utilized the expression of the H2B-mCherry reporter to separate between mouse and APM cells. Remarkably, we detected expression of the H2B-mCherry reporter in cell populations identified as satellite cells and mesenchymal progenitors, indicating that APM-iPSCs preferentially contributed to these cell types (Figures 5G–5I, S7D, and S7E). However, we cannot rule out the possibility that APM cells are present in cell populations that do not express the H2B-mCherry reporter or that limited sequencing depth prevented the detection of all H2B-mCherry transcripts.
Lastly, we FACS-purified ITGA7^+^/mCHERRY^+^ cells and ITGA7^+^/mCHERRY^−^ satellite cells from several mouse-APM chimeras and established proliferating myogenic cell lines resembling myoblasts (Figure 5J). To assess whether the myoblasts carry differentiation capacities, we subjected them to a differentiation protocol that give rise to myotubes following serum and growth factor withdrawal (Figure S7F). This effort resulted in the formation of multinucleated mCHERRY^+^ myotubes from ITGA7^+^/mCHERRY^+^ APM myoblasts, and mCHERRY^−^ myotubes from ITGA7^+^/mCHERRY^−^ mouse myoblasts (Figures S7F and S7G). The myotubes also expressed myogenic differentiation and striation markers including MYHC and ACTN2 (Figures 5K and S7H). Importantly, the fusion index of APM and house mouse myoblasts was equivalent, ranging between 50% and 90% (Figure S7I). In summary, a postnatal transgenic ablation system of mouse PAX7^+^ cells substantially increased APM satellite cell formation in interspecies mouse-APM chimeras. These APM satellite cells gave rise to functional myoblast lines that differentiated into multinucleated myotubes in vitro.
Discussion
Evaluating the contribution of PSCs to full-term interspecies chimeras, beyond the house mouse and brown rat, has seldom been achieved. This contrasts with numerous studies documenting PSC contribution to interspecies embryonic chimeras,1^,^13 which include unique combinations such as mouse-naked mole rat, mouse-human, mouse-horse, mouse-bat, pig-human, monkey-human, and other combinations.10^,^15^,^32^,^52^,^53^,^54^,^55^,^56^,^57^,^58^,^59^,^60 One explanation for this observation is that a close evolutionary distance more readily enables the contribution of PSCs to full-term interspecies chimerism. Our study supports this idea, as iPSCs from the APM, which is evolutionarily closer to the house mouse (diverged 6.8 million years ago61) than to the brown rat (diverged 13.1 million years ago61), efficiently contributed to interspecies chimerism with mice without exhibiting noticeable developmental defects, unlike mouse-rat chimeras, which occasionally do.10^,^41^,^50^,^62^,^63
The inability to achieve full-term chimerism may also reflect limited potency of non-rodent iPSCs, rendering efforts to optimize reprogramming and culture conditions desirable. Indeed, modulation of signaling pathways can promote an EPSC or an intermediate PSC state, both of which enhance interspecies embryonic chimera formation.32^,^64 Similarly, the use of LCDM medium during APM reprogramming may have enhanced chimera competency, akin to how mouse iPSCs generated using 3 compounds promoted enhanced chimerism and tetraploid potency in mice.18 Furthermore, our use of an integrative reprogramming system that enables tight transgene expression control may have improved iPSC chimeric contribution. Future comparisons of this system with non-integrative reprogramming approaches for assessing the chimeric competency of APM-iPSCs will be valuable.
In addition, comparing various culture conditions for APM-iPSCs to determine optimal ones for PSC maintenance and contribution to chimerism is warranted, particularly since a recent study proposed an alternative condition for establishing chimera-competent APM-iPSCs.16 Our study expands upon this work through several notable findings including (1) identifying improved conditions for inducing pluripotency and gene editing in both male and female APM cells and generating APM-iPSCs carrying transgenic reporters; (2) demonstrating the formation of APM gastruloids that exhibit symmetry breaking; (3) reporting contributions to interspecies chimerism in internal organs and correlating chimerism levels with organ size; (4) identifying APM-iPSC contribution to the male germ line in an interspecies host; and (5) demonstrating preferential generation of APM satellite cells in interspecies chimeras through blastocyst complementation. Collectively, we believe the two studies support each other’s conclusions regarding APM-iPSCs and their in vivo differentiation potential.
Several additional avenues of investigation emanate from our study. For example, although APM-iPSCs contributed to testicular germ cells, it will be important to determine if these cells are functional, or partially impaired as previously reported for xenogeneic PSC-derived germ cells generated in interspecies mouse or rat hosts.41^,^65^,^66 It will also be of interest to assess whether sterile rodent hosts can enable exclusive contribution of APM-iPSCs to the germline and whether xenogeneic female germ cells can be generated in interspecies chimeras. While germline contribution is one of the highly stringent criteria to showcase pluripotency, the most stringent criterion is tetraploid complementation, which has solely been reported for mouse and rat PSCs.67^,^68 It will be of interest to attempt this assay using APM-iPSCs, once reproductive and transgenic techniques are established for this species. Furthermore, considering that APM is one of the smallest mammalian species, it will be interesting to investigate if blastocyst complementation of APM-iPSCs and larger rodent hosts can give rise to xenogeneic APM organs such as the brain or pancreas of the size of the recipient host, similar to how a mouse PSC-derived pancreas of a rat size was generated in a Pdx1-knockout (KO) rat-mouse chimera.11
Finally, the APM is a unique mammal due to its diverse karyotypic evolution (2n = 18–34 depending on the clade) and intriguing reproductive biology, characterized by naturally occurring X∗Y sex-reversed females. This phenomenon represents an unusual evolutionary sex specification strategy in eutherian mammals.22^,^35^,^69 Since PSCs serve as a powerful tool to investigate developmental questions, we anticipate that establishing the APM-iPSC model will aid in elucidating the reproductive biology of this mammal during embryonic development, shedding light on its unusual evolutionary adaptation. Specifically, this model could facilitate the identification of the hypothesized demasculinizing factor located on the X∗ chromosome in X∗Y females, thereby providing insights into the rare sex determination strategy of this species.
Limitations of the study
Our study used predominantly male APM-iPSCs to evaluate chimerism and blastocyst complementation. It will be important to investigate these models using female APM-iPSCs to confirm whether our conclusions hold true for both sexes. Furthermore, the observed trend toward decreased organ weight could be explored by increasing the sample size of analyzed chimeras, as both the degree of chimerism and the preferential contribution of donor cells to specific organs add variability to statistical evaluation. In relation to this, interpretation of increased contributions to specific organs may also depend on the iPSC clone used, suggesting that future studies should incorporate additional APM-iPSC clones to assess the reproducibility of chimerism and organ-specific contribution patterns. Regarding blastocyst complementation, we explored whether APM-iPSCs could reconstitute a vacant tissue niche in the Pax7*^Cre^^/^^ERT2^ × R26**^LSL^**^-^^DTA^ model postnatally. Given that Pax7 is expressed during early vertebrate and neural embryonic development and that skeletal muscle growth continues from birth to adulthood, it would be of interest to examine whether APM-iPSCs perform better in a Pax7-*KO model during embryogenesis.
Resource availability
Lead contact
Further information and requests for resources and reagents should be directed to the lead contact, Ori Bar-Nur ([email protected]).
Materials availability
The plasmids and additional specific materials reported in this study will be shared by the lead contact upon reasonable request.
Data and code availability
- •The scRNA-seq raw dataset can be accessed in the Gene Expression Omnibus (GEO) repository under accession number [GSE310034](GSE310034). All other data reported in this study will be shared by the lead contact upon reasonable request.
- •This paper does not report original code.
- •Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
Acknowledgments
The African pygmy mouse male cell line was held at the 10.13039/501100000831Natural History Museum, London (NHM), and originated from Professor Malcolm Ferguson-Smith, University of Cambridge. They were secured for future research and made available through the efforts of CryoArks, originally funded by 10.13039/501100000268BBSRC grant BB/R015260/1 (see https://www.cryoarks.org/). We would like to express our particular gratitude to the CryoArks loans committee and to Jackie Mackenzie-Dodds for coordinating the loan from NHM. We also wish to thank the CytoEvol facility of ISEM (Labex CEMEB) for the derivation of the female APM cell line. Additionally, we are grateful to Dr. Joel Zvick and Dr. Xhem Qabrati for their review of the manuscript and helpful suggestions and to Lucienne Maak for help with animal work. The graphical schematics were created with BioRender.com under a paid license. We acknowledge the use of a purchased African pygmy mouse image from iStock.com/GlobalP. Additionally, we used and adapted images, courtesy of NIAID BIOART (public domain): bioart.niaid.nih.gov/bioart/403,bioart.niaid.nih.gov/bioart/154, and bioart.niaid.nih.gov/bioart/40. We also acknowledge using the software Proofig for screening images.
Author contributions
P.G. and O.B.-N. conceptualized the experiments, interpreted the results, and wrote the manuscript. P.G. performed most experiments and analysis of results. A.L. performed most experiments and analyses involving the formation of APM satellite cells in interspecies chimeras, as well as generation and differentiation of myoblasts and their characterization. M.T.-S. helped with experiments involving chimera production and performed blastocyst injections and embryo transfers. D.T. oversaw the mouse and APM gastruloid generation and characterization. C.L.T. helped with isolation of dermal cells from interspecies chimeras and contributed to the generation of the scRNA-seq library and data analysis. J.A.d.S. oversaw the scRNA-seq analysis. N.B. helped with muscle sectioning and immunostaining. F.V. provided female APM fibroblasts, and A.S. oversaw and supervised the gastruloid formation assay. O.B.-N. supervised the study.
Declaration of interests
The authors declare no competing interests.
Declaration of generative AI and AI-assisted technologies in the writing process
The manuscript was written by the authors, who acknowledge the use of ChatGPT (OpenAI) to enhance clarity and identify spelling and grammatical errors. All suggested changes were reviewed and revised as necessary, and the authors take full responsibility for the content of the publication.
STAR★Methods
Key resources table
REAGENT or RESOURCESOURCEIDENTIFIERAntibodiesAnti-Ly-6A/E (Sca-1) FITCThermo Fisher ScientificCat# 11-5981-82; RRID: AB_465333Anti-mouse Integrin alpha 7 Alexa Fluor 750R&D SystemsCat# FAB3518S; RRID: AB_3649446Anti-mouse CD45 Alexa Fluor 488BioLegendCat# 103121; RRID: AB_493532Anti-mouse CD31 Alexa Fluor 488BioLegendCat# 102414; RRID: AB_493408Anti-mouse/human CD15 (SSEA-1) FITCBioLegendCat# 125612; RRID: AB_2561707Mouse anti-Oct4Thermo Fisher ScientificCat# MA1-104; RRID: AB_2536771Rabbit anti-Sox2Thermo Fisher ScientificCat# 48–1400; RRID: AB_2533841Rabbit anti-Sox2Cell Signaling TechnologyCat# 3579; RRID: AB_2195767Rabbit anti-NanogAbcamCat# ab80892; RRID: AB_2150114Mouse anti-troponin T (cardiac/slow) supernatantDevelopmental Studies Hybridoma BankCat# ct3; RRID: AB_528495Mouse anti-NestinThermo Fisher ScientificCat# MA1-110; RRID: AB_2536821Rabbit anti-BrachyuryCell Signaling TechnologyCat# 81694; RRID: AB_2799983Goat anti-BrachyuryR&D SystemsCat# AF2085; RRID: AB_2200235Mouse anti-alpha-actinin (sarcomeric)Sigma-AldrichCat# A7811; RRID: AB_476766Mouse anti-Myosin heavy chainR&D SystemsCat# MAB4470; RRID: AB_1293549Rabbit anti-mouse LamininAbcamCat# ab11575; RRID: AB_298179Mouse anti-mouse Pax7R&D SystemsCat# MAB1675; RRID: AB_2159833Rabbit anti-Ddx4/VasaAbcamCat# ab13840; RRID: AB_443012PNA Alexa Fluor 488Thermo Fisher ScientificCat# L21409; RRID: AB_2315178Goat anti-Mouse IgG1 Alexa Fluor 488Thermo Fisher ScientificCat# A-21121; RRID: AB_2535764Goat anti-Mouse IgG1 Alexa Fluor 647Thermo Fisher ScientificCat# A-21240; RRID: AB_2535809Donkey anti-Rabbit IgG (H + L) Alexa Fluor 546Thermo Fisher ScientificCat# A10040; RRID: AB_2534016Goat anti-Mouse IgG2a Alexa Fluor 546Thermo Fisher ScientificCat# A-21133; RRID: AB_2535772Goat anti-Rabbit IgG (H + L) Alexa Fluor 488Thermo Fisher ScientificCat# A-11008; RRID: AB_143165Mouse Anti-Goat IgG (H + L) Alexa Fluor 647Jackson ImmunoResearch LabsCat# 205-605-108; RRID: AB_2339076Donkey Anti-Rabbit IgG H&L Alexa Fluor 488AbcamCat# ab150073; RRID: AB_2636877Goat anti-Mouse IgG2b Alexa Fluor 488Thermo Fisher ScientificCat# A-21141; RRID: AB_2535778Donkey anti-Rabbit IgG (H + L) Alexa Fluor 647Thermo Fisher ScientificCat# A-31573; RRID: AB_2536183Bacterial and virus strainsLV-TetO-rOKSMVectorBuilder, this paperVB180819-1041vbrLV-TetO-EF1α-rtTA3VectorBuilder, construct previously described in Domenig et al.70Addgene plasmid, Cat# 184379LV-EF1α-H2B-RFPVectorBuilder, this paperVB191001-1069dymChemicals, peptides, and recombinant proteinsDMEM, high glucose, pyruvateThermo Fisher ScientificCat# 41966029DMEM, high glucose, GlutaMAX SupplementThermo Fisher ScientificCat# 61965026DMEM, low glucose, pyruvateThermo Fisher ScientificCat# 31885023Knockout DMEMThermo Fisher ScientificCat# 10829018DMEM-F12Thermo Fisher ScientificCat# 11330057Ham’s F-10 Nutrient Mix, HEPESThermo Fisher ScientificCat# 22390025Neurobasal mediumThermo Fisher ScientificCat# 21103049IMDM+GlutamaxThermo Fisher ScientificCat# 31980030M2 mediumSigma-AldrichCat# M7167EmbryoMax KSOM Mouse Embryo MediaSigma-AldrichCat# MR-106-DEmbryoMax Advanced KSOM Embryo MediumSigma-AldrichCat# MR-101-DFetal Bovine SerumThermo Fisher ScientificCat# A5256701Embryonic Stem Cell FBS, qualified, US originThermo Fisher ScientificCat# 16141079Horse SerumThermo Fisher ScientificCat# 16050122Knockout Serum ReplacementThermo Fisher ScientificCat# 10828028N-2 SupplementThermo Fisher ScientificCat# 17502048B-27 SupplementThermo Fisher ScientificCat# 17504044GlutaMAXThermo Fisher ScientificCat# 35050038Non-essential amino acidsThermo Fisher ScientificCat# 11140035L-GlutamineThermo Fisher ScientificCat# A2916801Sodium Pyruvate (100mM)Thermo Fisher ScientificCat# 11360039Penicillin-streptomycinThermo Fisher ScientificCat# 15140122β-mercaptoethanolThermo Fisher ScientificCat# 21985023Ascorbic acidSigma-AldrichCat# A92902CHIR99021Sigma-AldrichCat# SML1046CHIR99021R&D SystemsCat# 4423PD0325901Selleck ChemicalsCat# S1036PD0325901TocrisCat# 4192RepSoxR&D systemsCat# 3742*(S)-(+)-Dimethindene maleateTocrisCat# 1425Minocycline hydrochlorideTocrisCat# 3268SB-590885Sigma-AldrichCat# SML0501L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (AA-2P)Sigma-AldrichCat# A8960Basic fibroblasts growth factorR&D SystemsCat# 233-FB-500Mouse LIFPolygene TransgenicsCat# PG-A1140-0100Doxycycline hyclateSigma-AldrichCat# D9891TamoxifenSigma-AldrichCat# T5648-1GCorn oilSigma-AldrichCat# C8267PMSGProSpecCat# HOR-272hCGProSpecCat# HOR-250MatrigelCorningCat# CLS354234PolybreneSigma-AldrichCat# TR-1003Lipofectamine 3000 Transfection ReagentThermo Fisher ScientificCat# L3000008KaryoMAX™ Colcemid™ SolutionThermo Fisher ScientificCat# 15212012Accutase Cell Detachment SolutionMilliporeCat# SCR005AccutaseInnovative Cell Technologies, Inc.Cat# AT-1040.05% trypsin-EDTAThermo Fisher ScientificCat# 25300054Mineral oilSigma-AldrichCat# M8410ACK lysis bufferThermo Fisher ScientificCat# A1049201Collagenase Type IIThermo Fisher ScientificCat# 17101015Dispase IIThermo Fisher ScientificCat# 17105041PBS, pH 7.4Thermo Fisher ScientificCat# 10010015HBSSThermo Fisher Scientific,Cat# 14025050Sodium CitrateSigma-AldrichCat# W302600Potassium chlorideSigma-AldrichCat# P9541Acetic acidCarl RothCat# 7332.1MethanolVWRCat# 20847.295DirectPCR Lysis ReagentViagen BiotechCat# 102-TBovine serum albumin (BSA)AppliChemCat# A1391DAPISigma-AldrichCat# MBD0015-1MLeBioscience™ Calcein Violet 450 AM Viability DyeThermo Fisher ScientificCat# 65-0854-39Fluoromount-G Mounting MediumThermo Fisher ScientificCat# 00-4958-02ProLong™ Gold Antifade MountantThermo Fisher ScientificCat# P36934ProLong™ Glass Antifade MountantThermo Fisher ScientificCat# P36980Critical commercial assaysKaryotype AnalysisCell Guidance System Ltd (Cambridge, UK)N/AP3 Primary Cell 4D-Nucleofector® X Kit LLonzaCat# V4XP-3024Qiagen Blood & Tissue kitQiagenCat# 69506Qiagen Gel Extraction KitQiagenCat# 28706Chromium Next GEM Single Cell 3′ Reagent Kits v3.1 (Dual Index, 10× Genomics platform)10× GenomicsCat# PN-1000268mNSET™ (Non-Surgical Embryo Transfer) Device for MiceParaTechsCat# 60010Leukocyte Alkaline Phosphatase KitSigma-AldrichCat# 86R-1KTDeposited dataMouse-APM chimera: scRNA-seq datasetsThis paperGEO: [GSE310034](GSE310034)Experimental models: Cell linesXY-APM fibroblastsCryoArks, National History Museum, London, UKsee acknowledgmentsXX∗-APM embryonic fibroblastsDescribed in Veyrunes et al.71see acknowledgmentsE14 mouse embryonic stem cells (ES-E14TG2a)ATCCCat# CRL-1821; RRID: CVCL_9108XY-APM-iPSCsThis paperN/AXX∗-APM-iPSCsThis paperN/ADR4 MEFsCell SystemCat# ASF-1002Experimental models: Organisms/strainsMouse: B6(C)/Rj-Tyr^c/c^Janvier LabsN/AMouse: Rj:SWISSJanvier LabsN/AMouse: B6.129P2-Gt(ROSA)26Sor^tm1(DTA)Lky^/JThe Jackson Laboratory009669Mouse: B6.Cg-Pax7^tm1(cre/ERT2)Gaka^/JThe Jackson Laboratory017763OligonucleotidesAPM Gh-F: 5′-GGC TAC AGG TAA GCA TGG GT-3′This paper, design based on Gh sequence identified in Matsuya et al.72N/AAPM Gh-R: 5′-CCA GGT TAG GGA CCC CGG TC-3′This paper, design based on Gh sequence identified in Matsuya et al.72N/AMouse Gh-F: 5′-GCA TGC GCA AAT CCC GCT GG-3′This paperN/AMouse Gh-R: 5′-GCA TAA CCC CAG GCT AGT CC-3′This paperN/AT4-gRNA: 5′-GGC CGC ACC CTT CTC CGG AG-3′Nakao et al.46N/AR26*-F1*:* 5′-CCA AAG TCG CTC TGA GTT GTT ATC-3′JAX protocol for stock number 009669JAX Primer 13840R26-R: 5′-GAG CGG GAG AAA TGG ATA TG-3′JAX protocol for stock number 009669JAX Primer 13841R26-APM-F1: 5′-CCA GAG TCG CTC TGA GTT GTT ATC-3′This paperN/AR26-F2: 5′-GGA AAA GTC TCC ACC GGA CG-3′This paperN/Aconstruct-R: 5′-CTA CTG CGC CCT ACA GAT C -3′Abe et al.45N/ARecombinant DNApMaster12Wu et al.36Addgene plasmid, Cat# 58527pSpCas9(BB)-2A-GFP (PX458)Ran et al.73Addgene plasmid, Cat# 48138R26-H2B-mCherry HR donor vectorAbe et al.45Addgene plasmid, Cat# 137928R26-H2B-EGFP HR donor vectorAbe et al.45Addgene plasmid, Cat# 137925Software and algorithmsNIS-Elements AR (v5.21.03)Nikon Instruments Inc.https://industry.nikon.com/en-gb/resource-centre/software/ZEN 2.6 liteCarl Zeiss Microscopy GmbHhttps://www.zeiss.com/microscopy/en/products/software/zeiss-zen.htmlGraphPad Prism 10GraphPadhttps://www.graphpad.com/scientific-software/prism/FlowJoBD FACS Aria IIhttps://flowjo.com/downloadClustal Omega Multiple Sequence Alignment (MSA)Madeira et al.74https://www.ebi.ac.uk/jdispatcher/msa/clustalonf-core/scrnaseq pipeline v3.0.0Peltzer et al.75https://zenodo.org/records/14360028Cell Ranger (v8; 10X Genomics)Zheng et al.76https://www.10xgenomics.com/cn/support/software/cell-ranger/latestSeurat (v5)Hao et al.77https://satijalab.org/seurat/OtherAggreWell™800 6-well plateSTEMCELL TechnologiesCat# 34825Low adhesion 96 well platesCorningCat# CLS7007-24EA
Experimental model and study participant details
Animals
All animals were housed in Allentown cages, at room temperature (RT), at a relative humidity of 50–60%, with ad libitum available food and water in a 12h light-dark cycle. The B6 Albino mouse strain (B6(C)/Rj-Tyr^c/c^) was purchased from Janvier Labs (France) and used for the creation of blastocysts. Furthermore, the following mouse strains were purchased from the Jackson Laboratory and were mated in our animal facility to produce blastocysts: B6.129P2-Gt(ROSA)26Sor^tm1(DTA)Lky^/J (Stock No 009669, here referred to as Rosa26^lsl-DTA^) and B6.Cg-Pax7^tm1(cre/ERT2)Gaka^/J (Stock No: 017763 here referred to as Pax7^Cre/ERT2^). Additionally, Rj:SWISS (Janvier Labs, France) mice were used as foster females which were mated with Rj:SWISS vasectomized males (Janvier Labs, France) to induce pseudopregnancy. Male and female chimeras and non-chimeric littermates were analyzed at 10–11 weeks of age. All experiments involving animals were approved by the Federal Food and Safety and Veterinary Office and the Cantonal Veterinary office in Zurich under the animal license numbers: ZH124/19, ZH002/22, and ZH032/23.
APM and mouse cell lines used in this study
Male APM fibroblasts were obtained from the CryoArks biobank under an approved loan. The cells were tested by IDEXX BioAnalytics for common pathogens including Corynebacterium bovis, Corynebacterium sp. (HAC2), Ectromelia, EDIM, Hantaan, K virus, LCMV, LDEV, MAV1, MAV2, mCMV, MHV, MNV, Mouse kidney parvovirus (MKPV), MPV, MTV, MVM, Mycoplasma pulmonis, Mycoplasma sp., Polyoma, PVM, REO3, Sendai, TMEV. The cells tested negative for all indicated pathogens. Female APM fibroblasts were provided by Dr. Frédéric Veyrunes and previously described.71 They were tested for Mycoplasma (LT07-318, Lonza). Please refer to the acknowledgments section for more information. The mouse E14 ESCs were obtained from ATCC (CRL-1821).
Method details
Cell culture
APM fibroblasts were cultured in “fibroblast growth medium” composed of 1:1 Knockout DMEM (Thermo Fisher Scientific, 10829018) and high glucose DMEM (Thermo Fisher Scientific, 41966029), supplemented with 10% Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, A5256701), 5% Knockout serum replacement (Thermo Fisher Scientific, 10828028), 1% non-essential amino acids (NEAA) (Thermo Fisher Scientific, 11140035), 1% GlutaMAX (Thermo Fisher Scientific, 35050038), 1% penicillin-streptomycin (P/S) (Thermo Fisher Scientific, 15140122), 0.05% β-mercaptoethanol (Thermo Fisher Scientific, 21985023), and 5ng/ml basic fibroblast growth factor (bFGF) (R&D Systems, 233-FB-500). Cells were cultured on 0.1% gelatin-coated culture dishes and cultured in low oxygen (5% O_2_, 5% CO_2_).
The conventional mouse ESC medium (“Serum+LIF”) used in reprogramming and maintenance of APM-iPSCs was composed of KO-DMEM supplemented with 15% FBS, 1% NEAA, 1% GlutaMAX, 1% P/S, 0.05% β-mercaptoethanol, and 1000U/ml mouse LIF (ESLIF) (PolyGene Transgenics, PG-A1140-0100). The “N2B27 medium” was composed of DMEM-F12 (Thermo Fisher Scientific, 11330057), mixed 1:1 with Neurobasal medium (Thermo Fisher scientific, 21103049), 0.5% GlutaMAX, 0.5xN2 and 0.5xB27 supplements (Thermo Fisher Scientific, 17502048 and 17504044) with 1000U/ml mouse LIF. The E14 ESCs were cultured on 0.1% gelatin-coated culture dishes in DMEM (Thermo Fisher Scientific, 61965026) supplemented with 10% ES-grade FBS (Thermo Fisher Scientific, 16141-079), 1% NEAA (Thermo Fisher Scientific, 11140035), 1% Sodium Pyruvate (100 mM) (Thermo Fisher Scientific, 11360039), 1% P/S (Thermo Fisher Scientific, 15140122), 0.2% β-mercaptoethanol (Thermo Fisher Scientific, 31350010), 3μM CHIR99021 (R&D Systems, 4423), 1μM PD0325901 (Selleck Chemicals, S1036), 100u/ml LIF (produced in the Sendoel lab).
Primary myoblasts were cultured on plates coated with Matrigel (Corning, CLS354234) in “myoblast medium” consisting of 1:1 DMEM and F-10 media (Thermo Fisher Scientific, 22390025) supplemented with 20% FBS, 10% horse serum (Thermo Fisher Scientific, 16050122), 1% P/S, and 10ng/ml bFGF (R&D Systems, 233-FB-500).
Lentiviral transduction
Approximately 500K APM fibroblasts were plated per 10cm dish and transduced with concentrated lentiviruses (produced by VectorBuilder) using 30μL of LV-TetO-rOKSM (2.23 × 10ˆ8 TU/ml) and 25μL LV-TetO-EF1α-rtTA3 (1.52 × 10^9^ TU/ml), which were added to 5mL fibroblast growth medium supplemented with 5μg/ml Polybrene (Sigma-Aldrich, TR-1003) for 24h. On the following day, cells were washed twice with PBS (Thermo Fisher Scientific, 10010015) to remove the remaining viral supernatant, and directly split for reprogramming.
Production of APM-iPSCs
Transduced APM fibroblasts were seeded onto in-house made γ-irradiated DR4 mouse feeders in 6-well plates.80K APM fibroblasts were seeded per well of a 6-well plate in “fibroblast growth medium” overnight in 5% O_2_ and 5% CO_2_ at 37°C. On the next day, the medium was changed to the respective reprogramming condition with or without 2μg/ml doxycycline (Sigma-Aldrich, D9891). For the episomal reprogramming, 100K APM fibroblasts were seeded per well of a 6 well plate coated with 0.1% gelatin. The cells were incubated overnight in “fibroblasts growth medium” and were transfected for two consecutive days with 2.5μg of the episomal vector pMaster12 (Addgene #58527) using the Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific, L3000008). At 24h post transfection, the medium was changed to Serum+LIF+ACR. The following small molecules were used in this study for reprogramming or maintenance of APM-iPSCs at the indicated concentrations: 50μg/ml ascorbic acid (Sigma-Aldrich, A92902), 3μM GSK3-inhibitor CHIR99021 (R&D Systems, 4423), 1μM TGF-β-inhibitor RepSox (R&D systems, 3742), 1μM MEK inhibitor PD0325901 (Tocris, 4192), 2μM (S)-(+)-Dimethindene maleate (Tocris, 1425), 2μM Minocycline hydrochloride (Tocris, 3268), and 0.5μM B-Raf inhibitor SB-590885 (Sigma-Aldrich, SML0501).
Production of mouse DR4 and APM feeders
APM fibroblasts and DR4 mouse embryonic fibroblasts (Cell System, ASF-1002) were expanded for several passages in fibroblast growth medium on 0.1% gelatin-coated 15cm dishes at 37°C in low oxygen conditions (5% O_2_ and 5% CO_2_). On the next day, cells were harvested and γ-irradiated using an RS2000 irradiator (Rad Source).
Karyotype analysis
APM-iPSCs were passaged for 48h before karyotype analysis was performed. On the following day, KaryoMAX Colcemid Solution (1:1000, Thermo Fisher Scientific, 15212012) was added to the culture medium and the cells were incubated for 4-5h. Afterward, cells were dissociated using Accutase Cell Detachment Solution (Millipore, SCR005) for 4-5min at 37°C, 5% CO_2_. A single cell suspension was obtained by pipetting gently up and down. Cells were pelleted at 130 x g for 5min and resuspended in 37°C pre-warmed 4-5mL hypotonic solution, which was prepared by dissolving 0.5g Sodium Citrate (Sigma-Aldrich, W302600) + 0.56g potassium chloride (Sigma-Aldrich, P9541) in 200mL distilled water followed by an incubation step at RT for 30min. Subsequently, cells were centrifuged at 130 x g for 5min and fixed using a 1:3 solution of acetic acid (Carl Roth, 7332.1) and methanol (VWR, 20847.295), respectively. Chromosome spreads were analyzed by Cell Guidance System Ltd (Cambridge, UK).
Differentiation of APM-iPSCs into cardiomyocytes and neuronal cells
For embryoid body (EB) formation, APM-iPSCs were pre-plated on gelatin-coated dishes for 1.5h to remove feeder cells. Afterward, the supernatant was collected, and the cells were pelleted at 130 x g for 5min. Cells were then resuspended and ∼12 × 106 APM-iPSCs were seeded onto a well of an AggreWell800 6-well plate, according to the manufacturer`s protocol (STEMCELL Technologies, 34825) in 5mL induction medium for cardiomyocyte or neural differentiation. The induction medium for the differentiation into cardiomyocytes was described before for rat cardiomyocytes,37 consisting of IMDM+Glutamax (Thermo Fisher Scientific, 31980030), supplemented with 15% FBS, 1% NEAA, 0.2mM L-Glutamine (Thermo Fisher Scientific, A2916801), and 0.05% β-mercaptoethanol. The neural induction medium consisted of DMEM-F12 supplemented with 15% FBS, 1% NEAA, 1% P/S and 0.05% β-mercaptoethanol. After 2 days in induction medium, EBs formed and were then plated onto 0.1% gelatin-coated culture dishes in differentiation medium. To this end, the cardiomyocyte induction medium was supplemented with 100μM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (AA-2P, Sigma-Aldrich, A8960). For neural differentiation, the medium was changed to “N2B27 medium”. EBs were harvested and plated onto 0.1% gelatin-coated culture dishes and differentiated for 8–10 days. The medium was changed every other day.
Differentiation of APM-iPSCs and mouse E14 ESCs to gastruloids
Mouse and APM gastruloids were generated as previously described.78 In brief, ESCs or APM-iPSCs were washed twice with PBS and dissociated into single cells using Accutase (Innovative Cell Technologies, Inc., AT-104) for 4min at 37°C, in 5% CO_2_. The detached cells were collected in 4.5mL of E14 ESC medium containing 10% FBS, centrifuged at 200 x g for 4min at 4°C and washed once more with 10mL PBS. After a second centrifugation, the pellet was resuspended in 1mL of N2B27 medium, and the cell concentration was determined using a hemocytometer. Aggregation was initiated by seeding 400 cells in a 40μL of N2B27 medium into low adhesion 96 well plates (Corning, CLS7007-24EA) followed by incubation for 48h at 37°C, 5% CO_2_. At 48 h post-aggregation, 150μL of N2B27 supplemented with 3μM CHIR99021 (Sigma-Aldrich, SML1046) was added to each well. The medium was subsequently replaced every 24 h with fresh N2B27 lacking CHIR99021 until 120h after aggregation.
Myoblast culture and differentiation into myotubes
For myoblast differentiation, around 40K myoblasts were seeded onto Matrigel-coated 12-well plates in “myoblast medium”. The next day, the medium was changed to “differentiation medium” consisting of DMEM (Thermo Fisher Scientific, 41966029) supplemented with 2% horse serum (Thermo Fisher Scientific, 16050122), and 1% P/S. Cells were kept in the ”differentiation medium” for 5 days. The medium was changed every other day.
Lentiviral labeling of APM-iPSCs with constitutive H2B-RFP reporter
APM-iPSCs were dissociated with 0.05% trypsin-EDTA (Thermo Fisher Scientific, 25300054), which was followed by transduction with concentrated LV-EF1α-H2B-RFP lentiviruses for 1h in suspension. Subsequently, cells were centrifuged at 130 x g to remove the remaining viruses and seeded onto DR4 mouse feeders. At 5 days after transduction, RFP^+^APM-iPSCs were FACS-purified to obtain a homogenous cell line expressing H2B-RFP.
Targeting of the Rosa26 locus in APM-iPSCs
In order to identify the APM Rosa26 locus, the sequence of the Mus musculus Exon1-Intron1 of the locus (ENSMUST00000124246.6) was blasted against the genomic Mus minutoides assembly sequence (https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_902729485.2/). We identified a Mus musculus reported “T4-gRNA” sequence (5′-GGCCGCACCCTTCTCCGGAG-3′),46 which was found to be conserved in the Mus minutoides genome. The T4-gRNA sequence was cloned using Golden Gate Assembly into the PX458 Cas9 backbone plasmid (Addgene #48138) and used together with the previously reported R26-H2B-mCherry (Addgene #137928) or R26-H2B-EGFP (Addgene #137925) HR donor vector for targeting. The iPSCs were targeted with the Amaxa 4D-Nucleofector (Lonza) and the P3 Primary Cell 4D-Nucleofector X Kit L (V4XP-3024) according to the manufacturer’s protocol. In brief, 3.5 × 10^6^ APM-iPSCs were resuspended in 100μL P3 Primary Cell 4D-Nucleofector X Solution and electroporated using 12μg of donor vector and 3μg sgRNA vector (Pulse Code: CG-104). Subsequently, the cells were plated onto three 6-well plates with feeder cells in Serum+LIF+2i medium. Successfully targeted, mCHERRY^+^ colonies were expanded by picking single colonies either before or after FACS purification.
Polymerase chain reaction (PCR), Sanger and Oxford Nanopore sequencing
DNA was isolated from cell pellets using either a Qiagen Blood & Tissue kit (Qiagen, 69506) or lysed with DirectPCR Lysis Reagent (Viagen Biotech, 102-T) according to the manufacturer’s protocols. APM growth hormone (Gh) primers (APM *Gh-*F and APM *Gh-*R) were designed based on the previously identified growth hormone sequence.72 The commonly used Rosa26 primer pair (R26-F1 and R26-R) was used as previously described (The Jackson Laboratory, protocol for stock number 009669, Primer 13840 and 13841). Alternatively in Figures S4E and S6I, an optimized APM primer “*R26-*APM-F1” (A→G) was used together with R26-R. The 5′ junction PCR was performed using a primer *R26-*F2, binding outside the homology arm together with a primer “construct-R”45 binding the insert. PCR products were loaded onto a 1.2% Agarose gel (BioConcept, 7-01P02-R), separated via electrophoresis at 120 V for 1–1.5 h, extracted using the Qiagen Gel Extraction Kit (Qiagen, #28706) and subjected to Sanger or Oxford Nanopore Sequencing (Microsynth AG, Switzerland). The sequences were aligned using Clustal Omega Multiple Sequence Alignment (MSA) tool available at the EMBL-EBI web server.74
Tamoxifen administration
Tamoxifen (Sigma-Aldrich, T5648-1G) was reconstituted in corn oil (Sigma-Aldrich, C8267) and injected intraperitoneally (i.p.) into Pax7^Cre/ERT2^; Rosa26^LSL-DTA^ mice for ablation of PAX7 expressing cells. A 25μL of a 2mg/mL solution was injected postnatally between day 3–5 and once per week from day P10 until the animals were euthanized. After weaning, tamoxifen concentration was changed to 75mg tamoxifen/kg animal body weight.
Satellite cell isolation
Satellite cells were isolated as previously described.50 In brief, skeletal muscles were harvested, minced, centrifuged at 350g for 3min, and digested in DMEM solution (41966029, Thermo Fisher Scientific) containing 2mg/ml Collagenase Type II (Thermo Fisher Scientific, 17101015) for 90min at 37°C in a shaking water bath. A second digestion step followed using 0.2% Collagenase Type II (Thermo Fisher Scientific, 17101015), 0.4% Dispase II (Thermo Fisher Scientific, 17105041) in F-10 medium (Thermo Fisher Scientific, 22390025) containing 5.5% DMEM (Thermo Fisher Scientific, 41966029), 7% PBS, and 10% horse serum (Thermo Fisher Scientific, 16050122) for 30min. After digestion, an 18-gauge needle was used to dislodge the cells from the muscle fibers followed by 3 filtration steps with a 100μm, 70μm and 40μm cell strainers. The cells were then centrifuged, resuspended in FACS buffer (2% FBS in PBS) and kept on ice for surface marker-based satellite cell analysis and sorting. For satellite cell FACS-purification, cells were stained with conjugated anti-mouse LY-6A/E (SCA1) (1:100, Thermo Fisher, 11-5981-82), anti-mouse ITGA7 (1:100, R&D Systems, FAB3518S), anti-mouse CD45 (1:100, Biogend, 103121), anti-mouse PECAM1/CD31 (1:100, BioLegend, 102414), and DAPI (1:1000, Sigma-Aldrich MBD0015-1ML).
Single cell RNA sequencing
Single cell RNA sequencing (scRNA-Seq) was performed as previously described.50 Skeletal muscles were isolated, and 1g of muscle was homogenized, resuspended and digested in HBSS (Thermo Fisher Scientific, 14025050), containing 1.5% bovine serum albumin (BSA; AppliChem, A1391) and 0.2% Collagenase II (Thermo Fisher Scientific, 17101015) for 60-90min at 37°C in a shaking water bath. After digestion, medium consisting of low glucose DMEM (Thermo Fisher Scientific, 31885023) supplemented with 10% FBS was added to the sample. The sample was vortexed and filtered through a 100μm cell strainer. After straining, the sample was centrifuged at 350g for 5min, subsequently resuspended in 2mL ACK lysis buffer (Thermo Fisher Scientific, A1049201) and incubated on ice for 1min. After incubation, cold PBS was added to a final volume of 25mL. The sample was strained through a 40μm cell strainer and centrifuged for 8min at 650 x g. The pellet was resuspended in FACS buffer containing 0.5% BSA and placed on ice until a live staining was performed with eBioscience Calcein Violet 450 a.m. Viability Dye (1:10000, Thermo Fisher Scientific, 65-0854-39). Live cells were sorted, and 20K cells were used for the library preparation according to the manufacturer’s instruction of the Next GEM Single Cell 3′ Reagent Kits v3.1 (10X Genomics platform). Single cell sequencing of the libraries was performed by Novogene GmbH (Germany).
Analysis of scRNA-seq data
To facilitate discrimination between African Pygmy Mouse (APM) and Mus musculus cells, the full SA-H2B-mCherry-bGHpolyA transgene locus was appended to the mouse reference genome (GRCm39) as an additional chromosome. Single-cell RNA-seq reads from the chimeric muscle sample were aligned to this custom genome using the nf-core/scrnaseq pipeline v3.0.0.75 Cells with reads mapping to the mCherry sequence were classified as originating from the APM, whereas cells lacking mCherry expression were classified as Mus musculus. The resulting count matrices were imported into Seurat v577 for downstream analysis. Low-quality cells were filtered out based on the following criteria: cells with fewer than 250 or more than 5,000 detected RNA molecules, and cells with >5% of reads mapping to mitochondrial genes were removed. The top 2,000 most variable genes were identified using Seurat’s FindVariableFeatures function and used for principal component analysis (PCA) to reduce dimensionality. The first 13 principal components were used for clustering and t-distributed stochastic neighbor embedding (t-SNE) visualization. Differential expression analysis was performed using the FindAllMarkers function. Genes were considered significant markers if they had an average log2 fold change >1 and an adjusted p-value <0.05 (Bonferroni correction). Cell-type annotation was performed by comparing identified marker genes to known cell-type-specific markers.
Blastocyst injections, embryo transfer and assessment of chimerism
For blastocyst production, superovulation was induced in female mice via injection of 5IU PMSG (ProSpec, HOR-272) followed by, 48h later, an injection with 5IU hCG (ProSpec, HOR-250) and pairing to single-housed males. Embryos were flushed in M2 medium (Sigma-Aldrich, M7167), at the morula-stage. Alternatively, 24h before injections, B6 (B6(C)/Rj-Tyr^c/c^) albino embryos (Janvier Labs) were thawed at the morula-stage. Embryos were incubated overnight in EmbryoMax Advanced KSOM Embryo Medium (Sigma-Aldrich, MR-101-D) or EmbryoMax KSOM Mouse Embryo Media (Sigma-Aldrich, MR-106-D) at 37°C, 5% CO_2_. On the subsequent day, blastocyst injections were performed as previously described.41 APM-iPSCs cultured in “Serum+2i” were dissociated using Accutase Cell Detachment Solution (Millipore, SCR005) or 0.05% trypsin-EDTA (Thermo Fisher Scientific, 25300054), for 3-5min at 37°C, 5% CO_2_. Afterward, iPSCs were dissociated and plated for 1h on a 0.1% gelatin coated culture dish to remove feeder cells. Next, the supernatant was collected and centrifuged at 130 x g for 5min. The cell pellet was resuspended in conventional mouse ESC medium without inhibitors and kept on ice until injections. Around 10–15 iPSCs were injected into embryos. Between 10 and 17 injected blastocysts were transferred via non-surgical embryo transfer (ParaTechs, 60010) into foster females. Mouse-APM chimeras were identified by contribution to coat color chimerism or presence of fluorescent reporters.
Alkaline phosphatase staining
Cells were stained for Alkaline Phosphatase using a Leukocyte Alkaline Phosphatase Kit (Sigma-Aldrich, 86R-1KT) according to the manufacturer’s protocol with minor modifications. Specifically, cells were fixed and stained directly in tissue culture plates, applying the fixative and staining solutions as described in the protocol for glass slides.
Live SSEA1 immunofluorescence of APM-iPSCs
To stain live cultures of APM-iPSCs, FITC anti-mouse/human CD15 (SSEA1) antibody (1:20, BioLegend, 125612) was added directly to the culture medium of APM-iPSCs. Cells were incubated for 1.5h, then washed 2x with PBS before adding culture medium, followed by microscopy imaging for fluorescence.
Immunofluorescence staining of APM-iPSCs
APM-iPSCs, were washed 2x with PBS and fixed for 5min using 4% Paraformaldehyde (PFA, diluted in PBS to 4%, Fisher Scientific, 11400580). Next, cells were washed 2x with PBS and blocked/permeabilized with 2% bovine BSA (AppliChem, A1391) and 1% Triton X-100 (Sigma-Aldrich, X100-100ML) in PBS for 1h at RT. Primary and secondary antibodies were diluted in PBS containing 0.1% BSA. Cells were incubated with primary mouse anti-Oct4 (1:200, Thermo Fisher Scientific, MA1-104), rabbit anti-Sox2 (2.5μg/ml, Thermo Fisher Scientific 48–1400), or rabbit anti-Nanog (1:200, Abcam, ab80892) antibodies for 2.5h at RT. After washing the cells 2x with PBS, the cells were incubated with secondary anti-mouse IgG1 Alexa Fluor 488 antibody (1:400, Thermo Fisher Scientific, A-21121), anti-rabbit IgG Alexa Fluor 488, 647 or 546 antibody (1:400, Thermo Fisher Scientific, A-11008/A-31573/A10040) and DAPI (1:1000) for 1h at RT. Cells were washed twice with PBS and mounted using ProLong Gold Antifade Mountant (Thermo Fisher Scientific, P36934) before imaging.
Immunofluorescence of differentiated EBs
Differentiated EBs were washed 2x with PBS, fixed using 4% PFA for 5min, and washed 2x with PBS. Afterward, the cells were blocked/permeabilized in 2% BSA, 1% Triton in PBS for 1h at RT. To stain cardiomyocytes, primary mouse anti-Troponin T antibody (2μg/ml, DSHB, CT3) in PBS containing 2% BSA was added overnight at 4°C. The next day, cells were washed 2x with PBS, followed by incubation with secondary anti-mouse IgG2a Alexa Fluor 546 antibody (1:400, Thermo Fisher Scientific, A-21133) and DAPI (1:1000) for 1h at RT. Subsequently, the cells were washed 3x with PBS before immunofluorescence analysis. For cells that were stained for the neural marker Nestin, primary and secondary antibodies were diluted in 0.1% BSA in PBS. Cells were incubated with primary mouse anti-Nestin antibody (Thermo Fisher Scientific, MA1-110) for 3h at RT. Afterward, cells were washed 2x with PBS, and incubated with secondary anti-mouse IgG1 Alexa Fluor 647 (Thermo Fisher Scientific, A-21240) and DAPI (1:1000) for 1h at RT. Finally, cells were washed twice with PBS, mounted using ProLong Gold Antifade Mountant (Thermo Fisher Scientific, P36934) and analyzed.
Immunofluorescence of gastruloids
Gastruloids were stained as previously described with minor modifications.78 The gastruloids were harvested and washed with PBS before being fixed with 4% PFA for 24h. Gastruloids were then washed 3x with PBS. After 1 h of permeabilization in PBS-FT (PBS, 10% Fetal Bovine Serum, 0.2% Triton X-100) at RT, shaking gastruloids were incubated with primary anti- T/Brachyury (Cell Signaling, 81694) alone, or anti-Sox2 (Cell Signaling, 3579) co-stained with anti-T/Brachyury (R&D Systems, AF2085) over night at 4°C. The gastruloids were then washed 3x with PBS-FT before being incubated with a secondary antibody solution containing DAPI (1:1000) over night at 4°C. The following secondary antibodies were used: anti-rabbit IgG Alexa Fluor 488 (Thermo Fisher Scientific, A-11008) or anti-goat IgG Alexa Fluor 647 (Jackson ImmunoResearch, 205-605-108) co-stained with anti-rabbit IgG Alexa Fluor 488 (Abcam, ab150073). Gastruloids were either directly imaged in PBS or mounted in Fluoromount-G Mounting Medium (Thermo Fisher Scientific, 00-4958-02) on microscopy slides using double sided tape as spacer. For improved visualization the T/BRA signal in Figure S2 was pseudo-colored in violet.
Immunofluorescence staining of mouse and APM myotubes
Myotubes were fixed in 4% PFA for 5min at RT, washed 2x in PBS followed by blocking and permeabilization in blocking solution containing 2% BSA and 1% Triton X-100 for 30min. Cells were incubated with primary Anti-ACTN2 (1:750, Sigma-Aldrich, A7811) antibody or anti-MyHC (1:500, R&D Systems, MAB4470) diluted in blocking solution for 1h at RT. Following two washes with PBS, the cells were incubated with secondary anti-mouse IgG1 Alexa Fluor 488 (Thermo Fisher Scientific, A-21121) and/or anti-mouse IgG2b Alexa Fluor (1:250, Thermo Fisher Scientific, A-21141) antibodies as well as DAPI (Sigma-Aldrich, MBD0015-1ML, 1:1000) in blocking solution for 1h at RT. Cells were washed twice with PBS and mounted using ProLong Gold Antifade Mountant (Thermo Fisher Scientific, P36934).
Muscle freezing
Tibilias anterior (TA) muscles of chimeras were isolated, embedded, and frozen as previously described.50 Muscles were mounted on 10% Tragacanth (Sigma-Aldrich, G1128) in PBS placed on a wooden cork piece. Following a 30s pre-cooling step in isopentane in a glass beaker placed in liquid nitrogen, the samples were snap-frozen in liquid nitrogen and stored at −80°C. Using a cryostat (Leica, CM1950) sections of 10μm thickness were generated and placed on a glass side. The glass slides were stored at −80°C before immunofluorescence stainings.
Immunofluorescence staining of muscle cross-sections
As reported before,50 cryosection on microscopy slides were fixed in 4% PFA in PBS for 5min at RT, which was followed by two PBS washes. Blocking and staining were performed in the same solution consisting of 0.2% Triton X-100, and 1% BSA in PBS. After 15min blocking at RT, the slides were washed twice with PBS, and incubated with primary antibodies: Rabbit anti-mouse Laminin (1:100, Abcam, ab11575), and mouse anti-mouse PAX7 (1:100, R&D Systems, MAB1675) for 1h at RT. After subsequent two washes with PBS, the slides were incubated with the following secondary antibodies: Donkey anti-rabbit IgG 647 (1:250, Thermo Fisher Scientific, A-31573), goat anti-mouse IgG1 488 (Thermo Fisher Scientific, A-21121) and DAPI (1:1000) for 60min at RT. Following two final washes with PBS slides were mounted with a glass cover slide using ProLong Glass Antifade Mountant (Thermo Fisher Scientific, P36980). For improved visualization the Laminin (LAMA) signal was pseudo-colored in white using the NIS-Elements software.
Immunofluorescence staining of testes cross-sections
Testes were freshly fixed in 4% PFA overnight at 4°C, followed by an incubation in a 30% sucrose in PBS solution for 24 h at 4°C for cryoprotection. Fixed testes were embedded in OCT (CellPath, KMA-0100-00A) and frozen on dry ice before storage at −80°C. Staining of the sections was performed as previously described.41 Testes cross sections were washed three times with PBS before incubation in blocking/permeabilization solution consisting of 2% BSA and 0.1% Triton X-100 in PBS for 1h. After blocking, the testes were incubated with primary anti-VASA/DDX4 (1:200, Abcam, ab13840) and conjugated PNA Alexa Fluor 488 (1:400, Thermo Fisher Scientific, L21409) antibodies in the staining solution with 2% BSA in PBS overnight at 4°C. On the next day, cells were washed twice with PBS before incubation with a secondary donkey anti-rabbit IgG Alexa Fluor 647 antibody (1:400, Thermo Fisher Scientific, A-31573) and DAPI (1:1000, Sigma-Aldrich, MBD0015-1ML) in staining solution for 1h at RT. Sections were washed twice, and mounted with ProLong Glass Antifade Mountant (Thermo Fisher Scientific, P36980) before sealed with a coverslip. For improved visualization, the VASA signal was pseudo colored in green and the PNA in white using the NIS-Elements software.
Dermal cell isolation
Dermal cells from mouse-APM chimeras were isolated as described (CELLnTEC, protocols) incorporating modifications derived from previously reported protocols.79^,^80^,^81 First, the hair was trimmed with clippers, then shaving cream was applied to remove any remaining hair. The bare skin was then removed from the deceased animal and cleaned with Braunol (B.Braun, 18392) and 70% ethanol. Following three washes with PBS, the subcutaneous fat and connective tissue was removed from the skin. With a scalpel, the skin tissue was cut into small pieces, directly transferred into 2.5 U/ml Dispase II solution in PBS (Thermo Fisher Scientific, 17105041) and incubated overnight at 4°C. Next, epidermis and dermis were carefully separated while being submerged in “washing medium” DMEM (Thermo Fisher Scientific, 41966029) supplemented with 2% P/S (Thermo Fisher Scientific, 15140122). The dermis was collected in “washing medium” and centrifuged at 200 x g for 10min. Collagenase II solution consisting of DMEM supplemented with 200 U/mL Collagenase II (Thermo Fisher Scientific, 17101015) was added to the pellet and the sample was incubated at 37°C for 1–2 h in a water bath under mechanical shaking. The solution was filtered through a 70μm cell strainer (Falcon, FAL352350) and then centrifuged at 400 x g for 5min at RT. The cell pellet was resuspended in “fibroblast growth medium” and incubated at 37°C, 5% CO_2._
FACS-purification of fluorescently labeled APM cells
RFP^+^ or mCHERRY^+^ APM cells were dissociated from plates and then centrifuged for 5min at 130 x g for iPSCs or 300 x g for dermal cells. The pellet was resuspended in PBS containing 2% BSA, filtered through a 40μM cell strainer and stained with DAPI (Sigma-Aldrich, MBD0015-1ML, 1:1000). FACS-purification of RFP^+^ or mCHERRY^+^ cell populations was performed using a Sony SH800S Cell Sorter (Sony Biotechnology Inc.). Analysis was performed using the software FlowJo 10.
Organ and embryo isolation and visualization
Organs were harvested immediately after euthanasia of the animals. Images of animals were taken with a Canon PowerShot G7 X Mark II camera or an iPhone 14 Pro. Organs and E11.5 embryos were harvested from the animals and rinsed in ice-cold PBS before imaging with a Nikon SMZ-1270 stereomicroscope with a QImaging Retiga R1 camera. The RFP signal was pseudo-colored using the NIS-Elements Software. All microscopy images were taken with a Nikon eclipse Ti2-E microscope.
Quantification and statistical analysis
Statistical analysis was performed using GraphPad Prism 10. Endpoint body and organ weights were compared between control and chimera groups for males and females separately using unpaired two-tailed t-tests. Repeated body weight measurements were analyzed separately for males and females using a two-way repeated-measures ANOVA with the Geisser-Greenhouse correction and a Šídák’s multiple comparisons test was used to compare weekly body weight means between groups. All p-values are displayed to three decimal places without rounding.
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