This is the accepted version of the journal article: Ibáñez, Elena; Santaló, Josep; Ibáñez, Elena. «Efficient generation of embryonic stem cells from single blastomeres of cryopreserved mouse embryos in the presence of signalling modulators». Reproduction, Fertility and Development, Vol. 34, Num. 7 (February 2022), p. 576-587. DOI 10.1071/RD21297 This version is available at https://ddd.uab.cat/record/257939 under the terms of the license 1 Efficient generation of embryonic stem cells from single 2 blastomeres of cryopreserved mouse embryos in the presence 3 of signaling modulators 4 5 Sandra Alonso-Alonso1, Josep Santaló1, Elena Ibáñez1* 6 1 7 Departament de Biologia Cel·lular, Fisiologia i Immunologia, Facultat de Biociències, 8 Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain 9 10 *Corresponding author. Email: elena.ibanez@uab.cat 11 12 Abstract 13 Context: Derivation of embryonic stem cells (ESC) from single blastomeres is an interesting 14 alternative to the use of whole blastocysts. But derivation rates are lower and the 15 requirements for successful ESC obtention are still poorly defined. 16 Aims: To investigate the effects of embryo cryopreservation and of signaling modulators 17 present during embryo culture and/or ESC establishment on ESC derivation efficiency from 18 single 8-cell mouse blastomeres. 19 Method: Fresh and cryopreserved 2-cell embryos were cultured and biopsied at the 8-cell 20 stage. Single blastomeres were cultured in the presence of 2i or R2i cocktails, with or without 21 adrenocorticotropic hormone (ACTH). We analyzed ESC derivation efficiencies and 22 characterized pluripotency genes expression and karyotype integrity of the resulting lines. We 23 also evaluated the impact of embryo preculture with R2i on epiblast cell numbers and 24 derivation rates. 25 Key results: ESC generation was not compromised by embryo cryopreservation and ACTH was 26 dispensable under most of the conditions tested. While 2i and R2i were similarly effective for 27 ESC derivation, R2i provided higher karyotype integrity. Embryo preculture with R2i yielded 28 increased numbers of epiblast cells but did not lead to increased ESC generation. 29 Conclusions: Our findings help to define a simplified and efficient procedure for the 30 establishment of mouse ESC from single 8-cell blastomeres. 31 Implications: This study will contribute to improve the potential of this experimental 32 procedure, providing a tool to investigate the developmental potential of blastomeres isolated 1 33 from different embryonic stages and to reduce the number of embryos needed for ESC 34 derivation. 35 36 Keywords 37 Embryonic stem cells, single blastomeres, mouse embryos, TGBβ inhibition, ERK inhibition, 38 adrenocorticotropic hormone, embryo culture, embryo cryopreservation 2 39 Introduction 40 Embryonic stem cells (ESC) are pluripotent cells with infinite self-renewal ability in vitro and 41 able to generate all derivates of the three embryonic germ layers. These fundamental features 42 enable ESCs to be a powerful tool for basic and applied research in regenerative medicine, 43 development biology, disease modeling and drug testing (Avior et al., 2016; Mahla, 2016; 44 Weinberger et al., 2016). 45 Traditionally, ESC have been derived from the inner cell mass (ICM) of blastocysts (Bc-ESC) 46 (Evans and Kaufman, 1981; Martin, 1981), but they can also be established from earlier 47 embryonic stages (Strelchenko et al., 2004; Tesar, 2005) and even from single blastomeres 48 (Bm-ESC) (Delhaise et al., 1996; Klimanskaya et al., 2006; Chung et al., 2006; González et al., 49 2010). In humans, this latter approach could circumvent the ethical concerns associated with 50 embryo destruction and could potentially allow the generation of autologous ESC lines for 51 children born from embryos transferred in preimplantation genetic screening cycles 52 (Klimanskaya et al., 2006). However, the extremely low efficiencies of Bm-ESC derivation in 53 humans have so far precluded the translation of this approach into the clinical practice. A 54 more realistic application of Bm-ESC derivation is based on the separate use of all the 55 blastomeres from an 8-cell embryo to initiate the derivation process. This, though leading to 56 the destruction of the used embryos, could increase the probability of obtaining at least one 57 ESC line from each embryo, thus reducing the total number of embryos needed to generate 58 ESC lines both in clinical and research settings. On the other hand, the generation of Bm-ESC 59 offers a powerful tool to investigate the development potential of blastomeres isolated from 60 mammalian embryos at different developmental stages, and especially past the two-cell stage, 61 when the ability of all the blastomeres to develop autonomously into blastocysts and liveborn 62 animals is highly reduced (Boiani et al., 2019). Although these applications are feasible in the 63 mouse with the current efficiencies of Bm-ESC derivation, technical limitations still exist and 64 improvements in Bm-ESC derivation, particularly from 8-cell stage embryos, should be 65 implemented to achieve the full potential of this experimental approach. 66 In humans, hESC are typically derived from cryopreserved embryos, but this practice is 67 uncommon when working with mice, so the effects of cryopreservation on mESC derivation 68 efficiencies and on the characteristics of the lines produced are largely unknown. Nonetheless, 69 the use of cryopreserved embryos as the source of mESC could help to simplify and optimize 70 the procedure, particularly for the longer and more complex Bm-ESC derivation, involving an 71 extra step of embryo biopsy. So far, only one study has investigated the effects of embryo 3 72 cryopreservation on mESC derivation, and the authors reported that it probably affected 73 derivation efficiency, as well as the karyotype, pluripotency genes expression, and 74 differentiation capacity of the mESC lines produced from whole blastocysts (Assadollahi et al., 75 2019). Whether cryopreservation also affects Bm-ESC derivation is presently unknown. 76 Indeed, requirements for successful Bm-ESC derivation are not exactly the same as for ESC 77 derivation from whole embryos, particularly regarding culture conditions. For instance, the 78 adrenocorticotropic protein hormone (ACTH), a peptide hormone produced by the pituitary 79 gland and involved in the response to biological stress, is often used for Bm-ESC derivation, but 80 unnecessary for ESC derivation from blastocysts. ACTH has been shown to promote ESC 81 proliferation and to have a positive effect on single blastomere culture and Bm-ESC derivation 82 when present in the derivation medium until outgrowth formation (Ogawa et al., 2004; 83 Wakayama et al., 2007; Lorthongpanich et al., 2008; Lee et al., 2012). Nonetheless, some 84 mouse Bm-ESC lines have been obtained without ACTH supplementation (Hassani et al., 85 2014a), so it is unclear whether ACTH has an essential role in efficient Bm-ESC derivation, 86 especially from 8-cell stage embryos. 87 Other signaling modulators have been shown to promote both Bc-ESC and Bm-ESC derivation 88 when added to the culture media. Notably, the introduction of the 2i cocktail to mouse ESC 89 derivation medium (Ying et al., 2008) represented a significant advance in the derivation and 90 pluripotency maintenance of Bc-ESC and Bm-ESC. This cocktail consists of a combination of the 91 mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor PD0325901 and the glycogen 92 synthase kinase 3 (GSK3) inhibitor CHIR99021. In the case of Bm-ESC from 8-cell blastomeres, 93 derivation efficiencies up to 25% can be obtained when 2i is added to culture medium 94 containing Knockout Serum Replacement (KSR), Leukemia Inhibitory Factor (LIF) and ACTH (Lee 95 et al., 2012; Vila-Cejudo et al., 2019) or N2B27 medium containing LIF (Hassani et al., 2014a). 96 Alternatively, the use of the R2i cocktail, composed of PD0325901 and the transforming 97 growth factor β (TGFβ) inhibitor SB431542, has resulted in derivation rates of up to 50% from 98 single blastomeres of 8-cell embryos from the NMRI and BALB/c non-permissive strains in 99 N2B27/LIF (Hassani et al., 2014a). In addition to the increase in derivation rates, these two 100 cocktails maintain ESC in a ground state of pluripotency, characterized by the expression of 101 core (Oct4, Nanog, Sox2) and naïve (Rex1, Esrrb, Tfcp2l1, etc.) pluripotency markers. However, 102 in the case of the R2i cocktail, the number of 8-cell embryos used in the study was low (n=18) 103 and no other groups have replicated these experiments, so more research is needed to 104 validate the higher efficacy of R2i versus the 2i cocktail in terms of Bm-ESC derivation and to 4 105 test its potential benefits also in permissive mouse strains. The effects of the two cocktails on 106 Bm-ESC derivation from cryopreserved embryos also deserves investigation. 107 In this study, we aimed to simplify and improve Bm-ESC derivation by investigating the impact 108 of embryo cryopreservation and culture media supplements (ACTH, 2i, R2i) on Bm-ESC 109 derivation from single blastomeres biopsied from 8-cell mouse embryos from the permissive 110 B6CBAF1 strain. We analyze derivation efficiencies and characterize the expression profile of 111 pluripotency genes and the karyotype integrity of the resulting Bm-ESC lines. In addition, we 112 also evaluate the effect of the preculture of embryos with the inhibitor cocktails prior to ESC 113 derivation. 114 5 115 Materials and methods 116 Animal procedures 117 Mouse care and procedures were conducted according to the protocols approved by the Ethics 118 Committee on Animal and Human Research of the Universitat Autònoma de Barcelona and by 119 the Departament d’Agricultura, Ramaderia, Pesca i Alimentació of the Generalitat de 120 Catalunya (permit numbers 4090 and 9995, respectively). 121 Embryo obtention and cryopreservation 122 Mouse embryos were obtained from 6-12 weeks old B6CBAF1/J females (Charles River 123 Laboratories). Females were induced to superovulate by intraperitoneal injection of 5 IU 124 Pregnant Mare’s Serum Gonadotropin (Foligon) followed by the injection of 5 IU Human 125 Chorionic Gonatropin (Divasa-Farmavic) 48 h later, and were mated with B6CBAF1/J males. 126 Embryos were collected at the 2-cell stage by flushing the oviducts with HEPES-buffered KSOM 127 medium (H-KSOM) (Biggers et al., 2000) 48 h later, and were divided into two groups: fresh 128 embryos, which were immediately transferred to culture dishes, and cryopreserved embryos, 129 which were slow-frozen (Costa-Borges et al., 2009) and stored in liquid N2 at -196ºC for a 130 minimum of 1 month before thawing and culture. 131 Embryo culture 132 Fresh and thawed 2-cell embryos were cultured in equilibrated EmbryoMax® KSOM Medium 133 (Merck) covered with mineral oil (Sigma) at 37ºC and 5% CO2 until the 8-cell stage, when the 134 biopsy was performed. 135 In some experiments, embryo culture media was supplemented with either the 2i or the R2i 136 cocktail (preculture groups). The 2i cocktail was a combination of PD0325901 (Axon Medchem; 137 1 μM) and CHIR99021 (Axon Medchem; 3 μM), whereas R2i was a combination of PD0325901 138 and SB 431542 (Sigma, 10 µM). These embryos were either biopsied when reaching the 8-cell 139 stage, and blastomeres used for mESC derivation, or maintained in culture until they reached 140 the blastocyst stage, to monitor their development and perform cell counts. 141 Blastomeres isolation and culture for stem cells derivation 142 When reaching the 8-cell stage, embryos were biopsied by micromanipulation in PBS (Sigma) 143 supplemented with 1% bovine serum albumin (BSA; Sigma) to obtain single blastomeres. Using 144 a double pipette holder, the zona pellucida was drilled with a 10 µm diameter pipette 145 containing acidic Tyrode’s solution and, immediately, all the blastomeres were individually 6 146 aspirated with a 25 µm diameter biopsy pipette containing PBS with 1% BSA. Within each 147 experimental group, blastomeres isolated from different embryos were pooled and randomly 148 distributed among the different treatments. 149 The isolated blastomeres were individually transferred to 50 µl drops of mESC derivation 150 medium that contained a feeder layer of mitomycin C-inactivated human foreskin fibroblasts 151 (HFF-1; ATCC®SCRC-1041™), in a 60 mm Petri dish covered with mineral oil (Sigma). Culture 152 dishes were prepared prior to the biopsy by adding several 50 µl 0.2% gelatin (Sigma) drops on 153 a 60 mm Petri dish. After a 10 min incubation at 37ºC, gelatin was pipetted out and HFF 154 solution drops were deposited at the same place and were covered with mineral oil. 155 Bm-ESC lines were derived using either KSR medium (K-medium) or N2B27 medium (N- 156 medium). The K-medium consisted of DMEM supplemented with 100 μM 2-β mercaptoethanol 157 (Gibco), 1x non-essential amino acids (Gibco), 50 U/ml penicillin and 50 μg/ml streptomycin 158 (Gibco), 20% KSR (Life Technologies) and 103 U/ml LIF (Merk Millipore). The N-medium was 159 composed of a 1:1 mixture of DMEM-F12 (Gibco) and neurobasal medium plus 100x N2 160 (Gibco), 50x B27 (Gibco) and 1 mM L-glutamine (Gibco) and was supplemented with 50 μM 2-β 161 mercaptoethanol, 1x non-essential amino acids, 103 U/ml LIF, 50 U/ml penicillin and 50 μg/ml 162 streptomycin. Both media were also supplemented with the 2i or the R2i inhibitor cocktails. 163 Moreover, when indicated, 0.1 mg/ml ACTH (Prospec) were added during the first week of 164 culture. This resulted in four different treatment groups in terms of the signaling modulators 165 used: 2i, R2i, 2i+ACTH and R2i+ACTH. At least three replicates per treatment were performed, 166 with a total of 60-70 blastomeres seeded for each treatment in K-medium and around 50 167 blastomeres in N-medium. 168 Blastomeres were cultured at 37ºC and 5% CO2, changing the medium every 48 h for 7-10 days 169 until outgrowths were observed. After the first passage, outgrowths were cultured in 4-well 170 plates without ACTH and putative mESC lines were subcultured, using trypsin-EDTA (BioWest), 171 once or twice a week and maintained in culture for at least 6 passages. 172 Characterization of stem cell lines 173 At the seventh passage, the stemness and differentiation potential of all the putative mESC 174 lines were assessed by Alkaline Phosphatase Activity (ALP) staining and immunodetection of 175 pluripotency and differentiation markers. Lines that were positive for these analyses were 176 considered genuine mESC lines. 177 ALP activity was determined using a commercial kit (Sigma AB0300). Cells were fixed in 4% 178 paraformaldehyde (PFA; Sigma) for 1 min at room temperature (RT) and washed twice with 7 179 PBS. Then, samples were washed twice with a 1:1 mixture of 5-bromine-4-chloride-3-inodyl 180 phosphate and nitroblue tetrazolium and incubated for 10 min at RT in the dark. The 181 pluripotency of the putative mESC lines was determined by the blue color of the colonies. 182 For immunofluorescence, putative mESC lines cultured on HFFs on glass coverslips and lines 183 subjected to spontaneous in vitro differentiation, by culturing them in DMEM supplemented 184 with 10% fetal bovine serum (FBS; BioWest) in feeder-free conditions for 10 days, were fixed 185 to analyze their pluripotency and differentiation potential, respectively. Fixation consisted of 186 an incubation in 4% PFA during 20 min at RT and three washes with PBS for 5 min each at RT. 187 Samples were blocked and permeabilized with a PBS solution containing 0.2% sodium azide 188 (Sigma), 0.5% Triton X-100 (Sigma) and 3% goat serum (BioWest) for 30 min at 37ºC. Next, they 189 were incubated with primary antibodies overnight at 4ºC, washed three times with PBS for 5 190 min and incubated with secondary antibodies for 2 h at RT. Samples were washed again three 191 times with PBS for 5 min, and the nuclear material was counterstained with 10 μg/ml Hoechst 192 33258 (Molecular Probes, Invitrogen) diluted in Vectashield (Vector Laboratories). Finally, 193 samples were mounted and analyzed with an epifluorescence microscope (Olympus BX61) and 194 the Cytovision software (Applied Imaging, Inc). 195 Mouse monoclonal anti-Oct4 (Santa Cruz Sc-5279, 1:50 dilution), rabbit polyclonal anti-Nanog 196 (Abcam, ab80892, 1:100 dilution) and rabbit polyclonal anti-Sox2 (Merck Millipore, ab5603, 197 1:200 dilution) antibodies were used to detect pluripotency markers. Mouse monoclonal anti- 198 Tubulin β3 (Tuj1; Biolegend 801201, 1:500 dilution), mouse monoclonal anti-α smooth muscle 199 actin (αSMA; Sigma A5228, 1:200 dilution) and mouse monoclonal anti-alpha-fetoprotein (AFP; 200 R&D Systems MAB1368, 1:50 dilution) antibodies were used to detect ectoderm, mesoderm 201 and endoderm differentiation markers, respectively. Secondary antibodies were anti-mouse 202 IgG Alexa Fluor 488 (Molecular Probes - Invitrogen A-21200, 1:500 dilution) for Oct4, Tuj1, 203 αSMA and AFP, and anti-rabbit IgG Alexa Fluor 594 (Molecular Probes - Invitrogen A-11037, 204 1:500 dilution) for Sox2 and Nanog. All the antibodies were diluted in a PBS-based solution 205 containing 0.2% sodium azide, 0.1% Triton X-100 and 3% goat serum. 206 Chromosome number analysis 207 Mouse ESC growing in a 35 mm Petri dish were treated for 7 h at 37ºC with 10 µg/ml 208 Karyomax® Colcemid Solution (Gibco 15210-040). After trypsinization, resuspended cells were 209 centrifuged for 5 min at 340 g, washed with PBS and centrifuged again for 5 min. Then, the 210 samples were exposed to a 0.075M KCl hypotonic solution for 8 min at 37ºC, centrifuged for 211 10 min at 620 g and fixed in 3 methanol: 1 acetic acid solution. The cells suspension was 8 212 dropped on degreased slides with absolute methanol previously dried with a tissue for 213 chromosome spreading, aged during 1.5-2 h at 120ºC and then stained with 10 μg/ml Hoechst 214 33258 diluted in Vectashield. 215 Three mESC lines were analyzed for each treatment. Images of 30-40 metaphases per line 216 were taken in an epifluorescence microscope (Olympus BX61) with the Cytovision software 217 (Applied Imaging, Inc) and the number of chromosomes was counted using ImageJ software. 218 Counts of 40 chromosomes were considered as a normal euploid karyotype. 219 RNA extraction and qPCR 220 The expression of pluripotency genes (Oct4, Nanog, Rex1, Esrrb and Tfcp2l1) was assessed by 221 real time quantitative PCR (qPCR) on some randomly selected blastomere-derived mESC lines 222 (two lines per treatment). STO mouse fibroblast were used as a negative control. 223 Total RNA was extracted from trypsinized ESC cultures using Maxwell RSC SimplyRNA Tissue Kit 224 (Promega) and its concentration and quality were assessed using a Nanodrop 225 spectrophotometer (ThermoFisher). One microgram of the extracted RNA was used as a 226 template for the reverse transcriptase reaction using the iScript cDNA Synthesis Kit (Bio-Rad). 227 The qPCR reactions were performed using iTaq Universal SYBR Green Supermix (Bio-Rad), on a 228 CFX96 Real-Time System thermocycler (Bio-Rad). The amplification program consisted in a first 229 denaturation step of 3 min at 95°C followed by 40 cycles of 10 s at 95°C (denaturing) and 30 s 230 at 60°C (annealing and extension). Finally, to assess the specificity of the amplification product, 231 an additional thermal denaturizing cycle was performed to obtain the melt curve of the qPCR 232 products. 233 Validated commercial PrimePCR SYBR Green Assays (BioRad) for Oct4 (Pou5f1, 234 qMmuCED0046525), Rex1 (Zfp42, qMmuCID0008767), Nanog (qMmuCID0005399), Esrrb 235 (qMmuCED0039638) and Tfcp2l1 (qMmuCID0013329) were used to assess pluripotency. A no 236 template control (NTC) was added for each primer, all samples were tested in triplicate and 237 the results were normalized to Gapdh (qMmuCED0027497) expression. The cycle 238 quantification value (Cq-value) was determined for each sample with the BioRad CFX 239 MaestroTM 195 Software and relative expression was calculated using the ΔΔCq method. 240 Blastocysts cell counts 241 Some of the embryos precultured in the presence of 2i or R2i were maintained in culture until 242 they reached the blastocyst stage (72 h). Blastocysts were fixed in 4% PFA during 15 min at RT 243 and blocked with a PBS solution containing 1% BSA, 0.05% Tween 20 and 0.2% sodium azide 9 244 overnight at 4ºC. Fixed embryos were incubated with a mixture of mouse monoclonal anti- 245 Oct4 (Santa Cruz Sc-5279, 1:50 dilution) and rabbit polyclonal anti-Nanog (Abcam ab80892, 246 1:200 dilution) primary antibodies diluted in block solution during 1.5 h at RT. Then, embryos 247 were washed three times with block solution for 10 min and incubated 30 min at RT in the dark 248 with a mixture of anti-mouse IgG-AF488 (Molecular Probes A21200, 6 µg/ml) and anti-rabbit 249 IgG-AF594 (Molecular Probes A11037, 6 µg/ml) secondary antibodies. Finally, they were 250 washed again thrice with block solution for 10 min, counterstained for 10 min with 10 µg/ml 251 Hoechst 33528 diluted in block solution and mounted in 10 μg/ml Hoechst 33258 diluted in 252 Vectashield. The number of total cells (Hoechst staining), ICM cells (positive for Oct4) and 253 epiblast cells (positive for Nanog) were counted on an epifluorescence microscope (Olympus 254 BX61) with the Cytovision and ImageJ software. 255 In these experiments, around 70 two-cell embryos were cultured per treatment group, with at 256 least four replicates, and around 40 blastocysts per group were used for cell counts. 257 Statistical analysis 258 Embryo development, outgrowth formation and mESC derivation rates were analyzed with χ2 259 and Fisher exact test, using GraphPad Prism software. 260 Relative expression results from qPCR analyses were compared with an ANOVA test with the 261 Bonferroni correction using the CFX Maestro software (Bio-Rad). 262 The number of ICM, epiblast and total cells were compared by One-Way ANOVA with post hoc 263 Tukey test, using GraphPad Prism software. 264 In all statistical analyses, p < 0.05 was considered statistically significant. 265 10 266 Results 267 Effect of embryo cryopreservation, signaling modulators and culture medium on mESC 268 derivation from single blastomeres 269 In the first set of experiments, a total of 546 blastomeres isolated from 8-cell embryos (n=70) 270 were allocated to 8 different experimental groups, depending on the source of embryos (fresh 271 or cryopreserved) and the signaling modulators added to K-medium during mESC derivation (2i 272 or R2i with or without ACTH). In all the experimental groups, most of the outgrowths produced 273 progressed in culture to form mESC lines (Table 1), with no remarkable differences in colonies 274 morphology among them. All 144 mESC lines generated showed stem-cell like morphology 275 (rounded colonies with defined edges), ALP activity, were positive for pluripotency markers 276 (ALP, OCT4 and NANOG) and, after spontaneous differentiation, were positive for markers of 277 three germ layers (TUJ1, α-SMA and AFP) (Figure 1). 278 In blastomeres biopsied from fresh embryos, the inhibitor cocktails 2i and R2i resulted in 279 similar mESC derivation efficiencies, but addition of ACTH during the first week of culture 280 decreased efficiencies, especially with the R2i treatment (Table 1). In particular, a significant 281 reduction of more than 50% was observed in the R2i group when comparing derivation in the 282 presence or absence of ACTH. Surprisingly, the reverse was observed in blastomeres obtained 283 from cryopreserved embryos, where addition of ACTH during the first week of culture in the 284 presence of R2i significantly increased derivation efficiencies by more than 50% and yielded 285 the highest derivation rates of all experimental groups (38.9%). No effect of ACTH was 286 observed in the case of 2i treatment. As in blastomeres of fresh embryos, no significant 287 differences between 2i and R2i groups were detected in mESC derivation efficiencies from 288 blastomeres of cryopreserved embryos (Table 1). 289 The source of blastomeres did not have a remarkable effect on mESC derivation efficiencies 290 either. When comparing results for the same treatment groups between fresh and 291 cryopreserved blastomeres, significant differences were only detected in the case of R2i, both 292 in the presence or absence of ACTH. As mentioned, ACTH had a deleterious effect on mESC 293 derivation from R2i-treated blastomeres isolated from fresh embryos, but a beneficial effect 294 on their cryopreserved counterparts. 295 In a second set of experiments, mESC derivation was attempted using the same treatments but 296 in N-medium. Given the lack of significant differences between the two embryo sources, only 297 blastomeres obtained from cryopreserved embryos were used in these experiments. Although 298 the percentage of outgrowths formed was high, without significant differences among 11 299 treatments, massive differentiation occurred at low passages resulting in extremely low rates 300 of mESC derivation in this medium (Table 2). 301 Effect of embryo cryopreservation and signaling modulators on pluripotency gene expression 302 and karyotype integrity of blastomere-derived mESC lines 303 Aside from the analyses used to validate the stemness of all the mESC lines obtained, some 304 lines were subjected to further characterization to assess the effect of embryo 305 cryopreservation and signaling modulators used during derivation and culture on the 306 expression of pluripotency markers (Figure 2.A) and karyotype integrity (Figure 2.B). To 307 determine the effect of signaling modulators, we randomly selected two (for qPCR) or three 308 (for chromosome counts) mESC lines derived from single blastomeres of cryopreserved 309 embryos from all the treatment groups in K-medium (2i, R2i, 2i+ACTH and R2i+ACTH). An 310 equivalent number of lines derived from fresh embryos of the 2i+ACTH group in K-medium was 311 also included to assess the effect of embryo cryopreservation. 312 Equivalent levels of Esrrb, Nanog and Tfcp21l gene expression were found by qPCR among the 313 different groups of mESC lines (Figure 2.A). Although lines derived from fresh embryos under 314 2i+ACTH treatment showed higher levels of expression of Oct4 and Rex1, differences were only 315 significant when compared with lines obtained from cryopreserved embryos under R2i (for 316 Oct4 expression) and R2i+ACTH (for Rex1 expression). The lack of significant differences 317 between 2i and R2i groups, with or without ACTH, and between groups with or without ACTH, 318 either in the presence of 2i or R2i, for any of the genes indicates that signaling modulators 319 used during derivation and/or culture have no significant effect on pluripotency gene 320 expression of the mESC lines generated. Nor does embryo cryopreservation, as no differences 321 were detected for any of the genes between lines derived in 2i+ACTH conditions from either 322 fresh or cryopreserved embryos. For all the genes analyzed, STO feeder cells, used as a 323 negative control, displayed barely undetectable expression levels (data not shown). 324 Chromosome counts in mESC lines derived from cryopreserved embryos under R2i treatments, 325 with or without ACTH, showed a normal modal karyotype (40 acrocentric chromosomes) and 326 high percentages (58.3-71.0%) of euploid cells (Figure 2.B). By contrast, mESC lines derived 327 from cryopreserved embryos under 2i treatments presented more chromosomal 328 abnormalities. In particular, in the absence of ACTH one polyploid line and one line showing a 329 chromosomal fusion were observed, and in the presence of ACTH the lack of one chromosome 330 was detected in almost 50% of the cells in one of the three lines studied. These abnormalities 331 were not observed in the 2i +ACTH group derived from fresh embryos, since the three lines 12 332 analyzed presented a normal modal karyotype and high percentages of euploid cells (>60%). 333 To elucidate a potential effect of embryo cryopreservation on the karyotype integrity of the 334 resulting mESC lines, we decided to include a new experimental group: three lines derived 335 from fresh embryos under R2i + ACTH treatment. The results showed that while two lines 336 retained an euploid modal karyotype of 40 acrocentric chromosomes (with >60% of euploid 337 cells), one line suffered the loss of one chromosome. 338 Effect of signaling modulators in embryo culture medium on blastocyst development, cell 339 counts and mESC derivation 340 Two-cell embryos were thawed and cultured in the continuous presence of 2i or R2i, in parallel 341 to a control group, and fixed at the blastocyst stage. No significant differences in 8-cell embryo 342 formation or blastocyst development were observed among the three groups (Table 3). 343 However, immunodetection analyses (Figure 3.A) revealed a significantly decreased number of 344 total cells of the blastocysts in the 2i group (77.6 ± 2.0) when compared with the control and 345 the R2i groups (85.4 ± 2.3 and 85.6 ± 1.8, respectively) (Figure 3.B). While R2i had no effect on 346 total cell numbers, it significantly increased the numbers of both ICM (23.0 ± 0.8) and epiblast 347 cells (14.9 ± 0.7) when compared with the control (19.6 ± 0.6 and 10.3 ± 0.5, respectively) and 348 the 2i groups (20.1 ± 0.7 and 11.4 ± 0.5, respectively) (Figure 3.B). 349 To assess whether the increase in epiblast cells observed with R2i could translate into higher 350 mESC derivation rates from the treated embryos, thawed 2-cell embryos precultured in the 351 presence of R2i were biopsied at the 8-cell stage and individual blastomeres were seeded in K- 352 medium supplemented with LIF, R2i and ACTH. No significant differences in mESC derivation 353 rates from control and R2i precultured embryos were detected (Table 4). 354 13 355 Discussion 356 With the aim to simplify and improve Bm-ESC derivation from 8-cell stage mouse embryos, in 357 the present study we evaluated the effect of embryo cryopreservation, derivation medium and 358 signaling modulators on derivation efficiency, pluripotency gene expression and karyotype 359 integrity of Bm-ESC lines. Furthermore, blastocyst formation, blastocyst cell number and Bm- 360 ESC derivation were analyzed after embryo culture in the presence of signaling modulators. 361 Embryo cryopreservation is a common and well-established procedure in mice, as well as in 362 other species. Nonetheless, several studies have shown that it can induce alterations in 363 cytoskeletal organization, mitochondrial function, gene expression patterns, blastocyst cell 364 numbers and allocation of cells to the ICM and trophectoderm (TE), among others, thus 365 compromising the developmental potential of the thawed embryos (Sohn et al., 2002; Larman 366 et al., 2011; Shin et al., 2011; Dasiman et al., 2013). Because of this, embryo cryopreservation 367 could potentially impact subsequent ESC derivation from the thawed embryos and the 368 characteristics of the ESC lines established, an issue that has been scarcely investigated. In the 369 present study, blastomeres obtained from frozen-thawed embryos produced Bm-ESC lines 370 with similar efficiencies than blastomeres obtained from fresh embryos, and no significant 371 differences in terms of pluripotency gene expression or karyotype integrity were observed 372 between the Bm-ESC lines derived from both embryo sources. These results enable us to 373 uncouple ESC derivation from embryo collection, thus shortening and simplifying Bm-ESC 374 derivation experiments and optimizing the use of the embryo donor females. Moreover, by 375 using cryopreserved embryos, donor embryos are available for biopsy and Bm-ESC derivation 376 at any time and derivation experiments are not subjected to the availability of female mice, 377 superovulation schedules or superovulation success. 378 Our results differ from those observed in a previous work by Assadollahi et al (2019) that, to 379 the best of our knowledge, is the only other study on the effects of embryo cryopreservation 380 on mouse ESC derivation. In that study, C57BL/6 mouse embryos vitrified at the 8-cell stage 381 were used for Bc-ESC derivation in 3i + LIF medium. Despite the limitations of the study (only 5 382 cryopreserved embryos used and no fresh control group), the authors concluded that 383 cryopreservation seems to affect ESC derivation efficiency as well as the characteristics of the 384 generated lines, such as karyotype or expression of pluripotency genes. Dissimilarities in the 385 mouse strains, embryo stage at the time of cryopreservation, cryopreservation method, 386 number of embryos used, source of the ESC (whole blastocysts vs. single 8-cell blastomeres) 387 and derivation medium employed could explain the different outcomes of the two studies. 14 388 A recent study by Vila-Cejudo et al (2019) showed that the original classification of mouse 389 strains into permissive and non-permissive for Bc-ESC derivation (Kawase et al., 1994; Brook 390 and Gardner, 1997) also stands when ESC are derived from single blastomeres, and this study 391 further identified the hybrid B6CBAF1 strain as permissive for both Bc-ESC and Bm-ESC 392 derivation. But they also demonstrated that, contrary to what occurs during Bc-ESC derivation, 393 the effect of the embryos’ genetic background on Bm-ESC derivation is not abolished by the 394 addition of the 2i cocktail. R2i has been proposed as an alternative to the widely used 2i and 395 proved to be as efficient as 2i in the derivation of Bc-ESC from several refractory strains and in 396 the maintenance of these cells in a ground state of pluripotency (Hassani et al., 2012, 2014b). 397 R2i has also been used for ESC derivation from single blastomeres of mouse embryos at the 2-, 398 4- and 8-cell stages, but only from two non-permissive strains, resulting in approximately 2- 399 fold higher efficiencies of Bm-ESC derivation than when 2i was used (Hassani et al., 2014a). 400 Our results corroborate that R2i produces high ESC derivation efficiencies from single 401 blastomeres from 8-cell embryos also in a permissive strain, but we failed to detect significant 402 differences between 2i and R2i. Bm-ESC derivation rates in 2i (22.5%-31.3%) were similar or 403 slightly higher than those previously obtained by our group (22.9%; Vila-Cejudo et al., 2019) in 404 the same mouse strain and culture conditions (KSR+ACTH+LIF) and than those reported by 405 Hassani et al (2014a) in the non-permissive strains cultured in N2B27+LIF (23%-25%). 406 Nonetheless, the derivation efficiency in R2i was slightly lower in our study (38.9% in the most 407 efficient conditions) than the 46-50% obtained by Hassani et al (2014a). We tried to replicate 408 the culture conditions from this other study by using N2B27 medium but, in agreement with 409 previous results in our laboratory (Vila-Cejudo et al., 2019, 2020), we did not succeed in 410 establishing Bm-ESC lines in this medium or did so but with extremely low efficiencies (<4%) 411 both when using 2i as when using R2i. Thus, in our hands, ESC lines can only be efficiently 412 established from single blastomeres of 8-cell embryos in KSR+LIF+2i or KSR+LIF+R2i. 413 Bm-ESC lines derived under 2i and R2i displayed similar expression levels of core and naïve 414 pluripotency genes, but R2i resulted in a higher number of lines with an euploid karyotype. 415 These results are in agreement with those previously reported in Bc-ESC lines (Hassani et al., 416 2014b), suggesting that the effects of these inhibitor cocktails on pluripotency gene expression 417 and karyotype integrity is equivalent in Bc-Esc and Bm-ESC lines and across strains. The higher 418 number of euploid lines under the R2i treatment may be attributed to the absence of 419 CHIR99021 in this cocktail, as it has been reported that prolonged inhibition of GSK3 induces 420 chromosomal instability in cultured cells (Tighe et al., 2007). 15 421 On the other hand, since the report that ACTH supports proliferation of single mouse ESC cells 422 in KSR+LIF without loss of pluripotency (Ogawa et al., 2004), ACTH has been regularly added to 423 the culture medium for the derivation of mouse ESC from single blastomeres, both in the 424 absence or presence of 2i (Wakayama et al., 2007; Lorthongpanich et al., 2008; González et al., 425 2010; Lee et al., 2012; Vila-Cejudo et al., 2019). Some of these studies have proved that single 426 blastomeres from 2- and 4-cell stage mouse embryos produce higher rates of Bm-ESC in the 427 presence than in the absence of ACTH (Lee et al., 2012; Lorthongpanich et al., 2008). But no 428 true comparisons have been made with blastomeres from embryos at the 8-cell stage. Our 429 results demonstrate that, under 2i conditions, generation of ESC from single blastomeres of 8- 430 cell embryos is not significantly enhanced by the presence of ACTH. These results agree with 431 those obtained by Gonzalez et al (2010), though in that study the derivation medium lacked 2i 432 and culture conditions slightly differed between blastomeres cultured in the presence or 433 absence of ACTH. Concerning R2i, there are no reports on the combined effects of this 434 inhibitor cocktail and ACTH on ESC derivation. Our observation that ACTH induced a significant 435 decrease in Bm-ESC derivation rates in the R2i group when blastomeres were obtained from 436 fresh embryos suggests a deleterious effect of the R2i+ACTH combination. Interestingly, such a 437 deleterious effect was also observed in blastocyst formation from 2-cell mouse embryos 438 cultured in N2B27+LIF+R2i in the presence of ACTH (Hassani et al., 2014a). Surprisingly, when 439 blastomeres were obtained from cryopreserved embryos, we observed the opposite effect, as 440 Bm-ESC derivation in R2i conditions was significantly improved by the presence of ACTH. We 441 cannot provide an explanation for these apparently incoherent effects of ACTH in fresh and 442 cryopreserved embryos, and this issue deserves further investigation. In any case, our results 443 indicate that ACTH is dispensable for Bm-ESC generation from blastomeres of 8-cell embryos, 444 except when cryopreserved embryos are used as the source of blastomeres and derivation is 445 performed under R2i conditions. As expected, because ACTH was only present during the first 446 week of culture, no long-term effects of ACTH (either positive or negative) were detected in 447 pluripotency gene expression or karyotype integrity of the established Bm-ESC lines. 448 Last, to try to further improve Bm-ESC derivation rates, we precultured the embryos in the 449 presence of 2i or R2i cocktails from the 2-cell stage. Signaling pathways play a critical role in 450 the first two differentiation events during early embryo development: the segregation of the 451 ICM and TE and, subsequently, the segregation of the epiblast and hypoblast from the ICM. 452 Thus, modulation of the endogenous signaling pathways can alter these differentiation events 453 and shift the balance of cells allocated to each lineage, as demonstrated by increased epiblast 454 cells numbers in mouse and human embryos cultured in the presence of 2i (Nichols et al., 16 455 2009; Van Der Jeught et al., 2013). Contrary to these previous results, we did not observe a 456 significant increase in epiblast (Nanog positive) cells in the 2i-treated blastocysts. But embryo 457 preculture in R2i significantly increased both ICM (Oct positive) and epiblast cell numbers, in 458 line with the reported effects of SB431542 in human embryos (Van der Jeught et al., 2014). 459 Although epiblast cells are considered the progenitors of ESC (Boroviak et al., 2014) and 460 increased numbers of epiblast cells have been correlated with higher rates of Bc-ESC 461 derivation in some studies (Campbell et al., 2013), we did not find enhanced Bm-ESC 462 establishment from embryos precultured in R2i. Unlike what occurs when whole embryos are 463 used, successful derivation of Bm-ESC lines requires that individual blastomeres are able to 464 generate epiblast cells. So it is possible that some blastomeres do not generate epiblast cells 465 despite the R2i treatment or that they generate an insufficient number of epiblast cells to 466 support the establishment of an ESC line. 467 In conclusion, we have provided evidence that embryo cryopreservation does not compromise 468 the potential of single blastomeres for Bm-ESC generation, which allows to simplify and 469 optimize Bm-ESC derivation procedures by uncoupling embryo collection from ESC derivation. 470 Moreover, we have confirmed and extended previous results on the effectiveness of the R2i 471 cocktail for the successful derivation and karyotype integrity of Bm-ESC lines, by using embryos 472 from a permissive strain and a KSR-based medium. Finally, we have demonstrated that ACTH is 473 dispensable for Bm-ESC generation (except when cryopreserved embryos and R2i conditions 474 are used) and that embryo preculture with R2i results in increased numbers of epiblast cells 475 but does not lead to increased Bm-ESC generation. Altogether, the findings of the present 476 study help to define a simplified and efficient procedure for the establishment of mouse ESC 477 lines from single blastomeres of 8-cell embryos. This will contribute to achieve the full 478 potential of this experimental procedure. 479 17 480 Conflicts of interest 481 The authors declare no conflicts of interest. 482 483 Declaration of funding 484 This work has been partially supported by the 2017-SGR-503 project from the Generalitat de 485 Catalunya. SAA was beneficiary of a PIF-UAB fellowship. 486 487 Acknowledgements 488 We thank staff from Servei Estabulari from Universitat Autònoma de Barcelona for animal 489 care, Jonatan Lucas and Maria Jesús Álvarez for help with feeder cell culture and reagent 490 preparation, Salvador Bartolomé for his assistance and advice in the design of the RT-qPCR 491 experiments and Ot Massafret and Aida Blanes for their support in the laboratory work. 492 493 Data availability 494 Available upon request. 495 18 496 References 497 Assadollahi, V., Hassanzadeh, K., Abdi, M., Alasvand, M., Nasseri, S. and Fathi, F. (2019). Effect 498 of embryo cryopreservation on derivation efficiency, pluripotency, and differentiation 499 capacity of mouse embryonic stem cells. J. Cell. Physiol. 234, 21962–21972. doi: 500 10.1002/jcp.28759 501 Avior, Y., Sagi, I. and Benvenisty, N. (2016). Pluripotent stem cells in disease modelling and 502 drug discovery. Nat. Rev. Mol. 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Treatment Blastomeres % Outgrowth formation % mESC lines Embryo source group (n) (n) (n) 2i 67 32.8 (22)a 31.3 (21)a a R2i 71 38.0 (27) 33.8 (24)a Fresh a,b 2i + ACTH 71 28.2 (20) 22.5 (16)a,b b R2i + ACTH 71 16.9 (12) 14.1 (10)b 2i 61 24.6 (15)a,b 24.6 (15)a,b b R2i 61 19.7 (12) 16.4 (10)b Cryopreserved a,b 2i + ACTH 72 27.8 (20) 26.4 (19)a,b a R2i + ACTH 72 38.9 (28) 38.9 (28)a a-b 621 Different superscripts indicate statistically significant differences (p<0.05) among treatment groups 622 within the same embryo source group (χ2 and Fisher exact test). 623 624 Table 2. Outgrowth formation and derivation efficiencies of mESC lines established under 625 different treatments with signaling modulators in N-medium from single blastomeres of 8-cell 626 embryos obtained from cryopreserved 2-cell embryos. Treatment Blastomeres % Outgrowth formation % mESC lines group (n) (n) (n) 2i 46 28.3 (13) 2.2 (1) R2i 46 32.6 (15) 0 2i + ACTH 54 38.9 (21) 0 R2i + ACTH 54 40.7 (22) 3.7 (2) 627 No significant differences were found among groups. 628 629 Table 3. In vitro development of two-cell embryos cultured in the presence or absence of 630 signaling modulators. Treatment Embryos % 8-cell embryos % blastocysts group (n) (n) (n) Control 69 82.6 (57) 79.7 (55) 2i 70 77.1 (54) 72.9 (51) R2i 68 85.3 (58) 83.8 (57) 631 No significant differences were found among groups. 632 23 633 Table 4. Outgrowth formation and derivation efficiencies of mESC lines established from single 634 blastomeres of 8-cell stage embryos obtained from cryopreserved 2-cell embryos cultured 635 with R2i in K-medium. Treatment Blastomeres % Outgrowth formation % mESC lines group (n) (n) (n) Control 347 25.4 (88) 24.8 (86) R2i 344 29.4 (101) 29.1 (100) 636 No significant differences between groups. 637 24 638 Figure Legends 639 Figure 1. Morphology of blastomere-derived mESC lines and immunodetection of 640 pluripotency and differentiation markers. (a-c) Morphology of aggregate formation at day 3 641 of blastomere seeding (a), outgrowth obtained at day 9 (b), and colonies of a mESC line 642 cultured for 6 passages (c), showing stem cell-like morphology with defined edges. (d-f) mESC 643 colonies expressing pluripotency markers OCT4 (green, d), SOX2 (red, e) and NANOG (red, f). 644 (g) mESC colonies showing alkaline phosphatase (ALP) activity (blue staining). (h-j) mESC 645 colonies expressing ectoderm marker TUJ1 (green, h), mesoderm marker αSMA (green, i) and 646 endoderm marker AFP (green, j) after spontaneous differentiation. In all the 647 immunofluorescence images, nuclear material is counterstained with Hoechst (blue). All the 648 scale bars represent 50 µm. 649 Figure 2. Expression of pluripotency markers and karyotype integrity in mESC lines 650 established under different treatments with signaling modulators in K-medium from single 651 blastomeres of 8-cell embryos derived from fresh or cryopreserved 2-cell embryos (A) 652 Expression of pluripotency markers measured by qPCR. Gapdh was used for normalization of 653 cDNA amount. Data represents the mean of two biological and three technical replicates ± 654 SEM for each treatment group. Significant differences are denoted with *p≤ 0.05 (One-way 655 ANOVA with Bonferroni correction). (B) Stacked bar chart showing the percentage of cells with 656 a normal (black) or abnormal (grey) chromosome number for each treatment group. Three 657 mESC lines were analyzed for each treatment. Numbers on top of the bars indicate the modal 658 karyotype of each line. F, fresh embryos. C, cryopreserved embryos. 659 660 Figure 3. Total, ICM and epiblast cells counts in blastocysts obtained from two-cell embryos 661 cultured in the presence or absence of signaling modulators. (A). Epifluorescence raw and 662 merge images of blastocysts immunostained for OCT4 and NANOG and counterstained with 663 Hoechst. All the scale bars represent 60 µm. (B) Mean number ± SEM of total cells (Hoechst 664 positive), ICM cells (OCT4 positive) and epiblast cells (NANOG positive) in blastocysts. Each 665 grey dot represents the number of cells of a single blastocyst. a-bDifferent superscripts indicate 666 statistically significant differences among treatments (p<0.05; ANOVA). 25