After the treadmill test, the mice were sacrificed and weighted, and then the muscles were also isolated and weighted (Supplementary Table 2). Animal body weight was 23.27 to 29.94\u00a0g (Supplementary Table 2). No significant differences were found between all samples analyzed (Supplementary Fig.\u00a01A\u2013C). To assess whether hydrogel transplantation influenced the structure of regenerating muscles, we stained the histological sections obtained from them with HE and then CSA and the percentage of regenerating fibers, characterized by centrally located nuclei, was determined. We did not find any significant differences between all the muscle groups, i.e., acute injured muscles injected with different types of hydrogels (Fig.\u00a01<\/a>B\u2013D). Next, we analyzed muscle sections stained with Masson\u2019s trichrome to determine the level of fibrosis in all the indicated muscle types and again found no significant differences between them (Fig.\u00a01<\/a>E\u2013F). Altogether, the results obtained indicated that reconstruction of the injured muscle in the presence of hydrogels proceeded properly and resulted in a better treadmill test outcome compared to untreated muscles.<\/p>\n Expression of myogenesis, angiogenesis, and neurogenesis markers in regenerating muscles transplanted with hydrogels modified with IL4 or SDF1 mimicking peptides<\/p>\n In the next experiments, we analyzed the expression of selected myogenesis, angiogenesis, and neurogenesis markers in the following SCID muscle groups: intact, untreated, transplanted with R1, R4-IL4 or R1-SDF1 to determine whether the presence of hydrogel influenced muscle reconstruction, including its innervation and vascularization. As myogenic markers we analyzed three types of myosin heavy chains, i.e. Myh3, which is expressed in embryonic and fetal but also in regenerating fibers, as well as Myh2 and Myh7, which are present in more mature fast and slow muscle fibers. In addition, we analyzed Myog, which is necessary for the differentiation of myogenic cells, Dmd, which encodes a structural protein that protects the integrity of the muscle fiber membrane, and finally Pax7, a marker of SCs, crucial cells for muscle regenerative potential. Among the angiogenesis markers analyzed by us there were Vwf, Cd31, Vegfa, and Flt1, while the neurogenesis markers included Achr, Musk, Lrp4, Dok7, and Rapsn.<\/p>\n The results indicated that muscles injected with R4-IL4 were characterized by significantly higher levels of Myh7, Dmd, and Pax7 expression compared to muscles injected with unfunctionalized hydrogel, as well as intact muscles (Fig.\u00a02<\/a>A). In the case of Dmd mRNA, a significant difference was also found between the muscles injected with R4-IL4 and the injured untreated ones (Fig.\u00a02<\/a>A). The level of Dmd expression was also significantly higher in muscles injected with the R1-SDF1 hydrogel compared to intact and untreated muscles and muscles injected with the unfunctionalized hydrogel (Fig.\u00a02<\/a>A). These muscles, i.e., transplanted with R1-SDF1, were also characterized by significantly higher levels of Myog mRNA, compared to untreated ones, as well as Myh7 mRNA, compared to intact muscles and muscles injected with control hydrogel (Fig.\u00a02<\/a>A). Furthermore, both muscles injected with functionalized hydrogels were characterized by an elevated level of Myh3 expression compared to intact muscles, and in case of R1-SDF1 treated muscles\u2014also to untreated ones (Fig.\u00a02<\/a>A). Although the final conclusion needs to be confirmed by further protein analysis, the results obtained by us suggest that both groups of muscles injected with functionalized hydrogels, i.e., R1-SDF1 and R4-IL4, were characterized by enhanced levels of myogenesis markers. Also in the case of mRNAs encoding angiogenesis markers, we found a significantly higher level of them in muscles transplanted with functionalized hydrogels\u2014Vwf compared to all other muscle groups (i.e., intact, untreated, R1) and Vegfa and Flt1 compared to intact and untreated muscles (Fig.\u00a02<\/a>B). The expression of neurogenesis markers was also significantly elevated in muscles injected with functionalized hydrogels. Significantly higher levels of Dok7 and Lrp4 expression were found in such muscles compared to intact and untreated ones (Fig.\u00a02<\/a>C). In the case of muscles treated with R4-IL4, the level of Dok7 mRNA was higher than in muscles injected with other hydrogels and Lrp4 mRNA was elevated in muscles treated with functionalized hydrogels compared to those injected with R1 hydrogel (Fig.\u00a02<\/a>C). All of the results indicate that transplantation of each functionalized hydrogel was beneficial for skeletal muscle reconstruction, as it supported expression of mRNAs encoding factors indispensable for myogenesis, angiogenesis, and innervation, i.e., processes that are crucial for regaining proper muscle functioning. In the case of Myh2, Cd31, Achr, Musk, Rapsn, we did not find significant differences in their expression between analyzed muscle groups (Supplementary Fig.\u00a02A-C).<\/p>\n Fig. 2 Expression of selected markers of myogenesis, angiogenesis and neurogenesis in regenerating SCID muscles injected with hydrogels modified with IL4 or SDF1 mimicking peptides. (A) Myogenesis markers (Myh3, Myh7, Myog, Dmd, Pax7); (B) Angiogenesis markers (Vwf, Vegfa, Flt1); (C) Neurogenesis markers (Lrp4, Dok7). For (A\u2013C) mean RQ (2-\u0394\u0394CT) values with standard deviation of at least 3 separate biological samples (obtained from different animals with results presented as dots) are shown. Intact muscles served as a reference sample. *p\u2009\u2264\u20090.05, **p\u2009\u2264\u20090.01, ***p\u2009\u2264\u20090.001, ****p\u2009\u2264\u20090.0001.<\/p>\n Complex analysis of muscles transplanted with hydrogels and myoblasts derived from human induced pluripotent stem cells<\/p>\n In the next stage of our studies, muscles of SCID mice were injured with BaCl2 and then transplanted not only with hydrogels but also with myoblasts obtained from hiPSCs. Two lines of hiPSCs were used, i.e., hiPSC-DMD-GFP, synthesizing DYSTROPHIN linked to the GFP as well as hiPSC-LMN\u03b21-GFP, producing fluorescently tagged LAMIN\u03b21. As a result, two types of myoblasts were generated\u2014MB-DMD and MB-LMN\u03b21\u2014hereinafter called DMD and LMN\u03b21, respectively. Before transplantation experiments we have assessed the viability of cells suspended in hydrogel-saline solution for 30\u00a0min, that is, the same time when during in vivo experiments the cells were injected into the muscles, kept in the incubator, and additionally for 2\u00a0h and 24\u00a0h. The mean value of the results obtained for a cell viability was 97% for 30\u00a0min incubation, 80% for 2\u00a0h, and 40% for 24\u00a0h. This is consistent with comprehensive chemical analysis that indicates that the structure of the hydrogel is appropriate for cells and shows that the hydrogels themselves are not detrimental for the cells suspended in them (Dier\u017cy\u0144ska et al., in preparation). The drop in viability observed after 24\u00a0h probably resulted from the lack of nutrient delivery in hydrogel saline solution. This environment significantly differs from the in vivo condition to which hydrogel-transplanted cells are exposed at this time-point (24\u00a0h).<\/p>\n As before, during in vivo experiments intact muscles or injured but untreated muscles served as control. Again, 28\u00a0days after transplantation, we performed a treadmill test and found the highest running distance and time for mice whose muscles were injected with R1-SDF1 hydrogel and DMD cells (Supplementary Fig.\u00a03A). Significantly elevated values were also observed in the case of mice whose muscles were injected with control hydrogel and DMD cells, compared to those which muscles were not treated (Supplementary Fig.\u00a03A). Detailed analysis of muscle structure, including CSA assessment, revealed no significant differences between analyzed muscle groups, i.e., intact, untreated, transplanted with different types of hydrogels and DMD cells, transplanted with different types of hydrogels and LMN\u03b21 cells (Supplementary Fig.\u00a03B, 3C). Analysis of the percentage of fibers with centrally located nuclei as well as fibrosis area did not reveal any significant differences between all indicated muscle groups (Supplementary Fig.\u00a03D\u2013E). No differences were also found in body or muscle weight between all these animal groups (Supplementary Fig.\u00a03F\u2013H).<\/p>\n Analysis of the expression of myogenesis, angiogenesis and neurogenesis markers showed that muscles injected with each type of hydrogel and DMD cells were characterized by significantly higher level of Myh3 expression compared to other variants of the experiment (Fig.\u00a03<\/a>). All types of muscles transplanted with hydrogel and cells, regardless of their types, had a significantly elevated level of Dmd mRNA in comparison to intact and untreated muscles (Fig.\u00a03<\/a>). Additionally, a significantly higher level of Pax7 mRNA was observed in the muscles injected with R1 and DMD cells compared to all other muscle groups (Fig.\u00a03<\/a>). Elevated Pax7 expression was also found in muscles injected with R4-IL4 and DMD cells (Fig.\u00a03<\/a>). Both indicated muscle groups (that is, injected with the R1 or R4-IL4 hydrogel and DMD cells) were also characterized by significantly elevated level of Myh7 expression compared to other muscle groups (Fig.\u00a03<\/a>). For angiogenesis markers, the most profound effect was observed for muscles injected with R4-IL4 hydrogel and DMD cells. These muscles were characterized by elevated expression of Vegfa and Flt1 compared to all other muscles (Fig.\u00a04<\/a>). Furthermore, we also observed a significantly higher level of Vwf mRNA in the muscles injected with R4-IL4 and LMN\u03b21 cell injected muscles as compared to other muscle groups (Fig.\u00a04<\/a>). The most profound effect in terms of neurogenesis marker expression was again found in case of the muscles injected with R4-IL4 and DMD cells, which were characterized by a significantly higher level of Musk and Rapsn mRNAs compared to all other muscle groups (Fig.\u00a04<\/a>). Furthermore, such muscles, as well as muscles injected with R1-SDF and DMD cells, had a significantly elevated level of Achr mRNA compared to all other samples (Fig.\u00a04<\/a>). All muscle groups injected with hydrogels and DMD cells were also characterized by a higher level of Lrp4 expression compared to control muscles as well as those injected with hydrogels and LMN\u03b21 cells (Fig.\u00a04<\/a>). Furthermore, muscles injected with R4-IL4 and LMN\u03b21 cells were characterized by a significantly higher Dok7 mRNA level compared to untreated muscles, as well as other selected muscles (Fig.\u00a04<\/a>). Together, although the results obtained by us need to be further verified at the protein level, they suggest that the addition of hiPSC-derived myoblasts, with properties similar to the DMD myoblasts used in the current study, may support myogenesis, angiogenesis, and neurogenesis in regenerating muscles, also in the case of co-injection with unfunctionalized R1 hydrogel. This effect was visible when muscles transplanted with hydrogels and DMD cells were compared to intact or untreated muscles (Fig.\u00a03<\/a>, 4<\/a>) but also when they were compared to muscles injected only with hydrogels, however, with few exceptions (Supplementary Figs.\u00a04, 5). In the case of Vwf, its mRNA level in muscles injected with R4-IL4 and LMN\u03b21 cells was comparable to that observed in the case of muscles injected with hydrogel alone and in control muscles (i.e., intact and untreated; Supplementary Fig.\u00a05). Furthermore, Dok7 expression turned out to be lower in muscles injected with R4-IL4 and LMN\u03b21 cells than in muscles injected with each functionalized hydrogel alone (Supplementary Fig.\u00a05). Despite these few differences, muscles injected with hydrogels and iPSC-derived myoblasts, mostly DMD ones, were characterized by elevated levels of mRNAs encoding the myogenesis, angiogenesis, and neurogenesis markers indicated above compared to both control muscles and those injected with hydrogels alone. No significant differences in treadmill results, body or muscle weight, as well as fibrosis and percentage of fibers with centrally located nuclei were found between muscles injected with hydrogels alone or with hydrogels and cells (Supplementary Fig.\u00a06).<\/p>\n Fig. 3 Expression of selected myogenesis markers in regenerating SCID muscles injected with hydrogels modified with IL4 or SDF1 mimicking peptides and hiPSC-derived myoblasts. Mean RQ (2-\u0394\u0394CT) values with standard deviation from at least 3 separate biological samples (obtained from different animals with results presented as dots) are shown for selected myogenesis markers (Myh2, Myh3, Myh7, Myog, Dmd, Pax7). Intact muscles served as a reference sample. *p\u2009\u2264\u20090.05, **p\u2009\u2264\u20090.01, ***p\u2009\u2264\u20090.001, ****p\u2009\u2264\u20090.0001.<\/p>\n Fig. 4 Expression of selected markers of angiogenesis and neurogenesis in regenerating SCID muscles injected with hydrogels modified with IL4 or SDF1 mimicking peptides and hiPSC-derived myoblasts. (A) Angiogenesis markers (Vwf, Cd31, Vegfa, Flt1); (B) Neurogenesis markers (Achr, Musk, Lrp4, Dok7, Rapsn). The mean RQ (2-\u0394\u0394CT) values with standard deviation from at least 3 separate biological samples (obtained from different animals with results presented as dots) are shown. Intact muscles served as a reference sample. *p\u2009\u2264\u20090.05, **p\u2009\u2264\u20090.01, ***p\u2009\u2264\u20090.001, ****p\u2009\u2264\u20090.0001.<\/p>\n In addition to the analysis mentioned above, we also performed immunohistochemistry staining using anti-human nuclear antigen (HNA) antibody or anti-GFP antibody to follow the fate of human cells transplanted to mouse muscles. We also performed PCR analysis using probes for human GAPDH,\u00a0HPRT, and ACTB, and additionally for GFP. Surprisingly, although we used SCID mice, we have found only single cells detected by indicated antibodies, as well as extremely low levels or even absence of human transcripts or GFP in mouse muscles analyzed 28\u00a0days after hydrogel and cell transplantation. To address this issue, we have performed additional in vivo experiments in which muscles were collected and analyzed by PCR at day 0, 3, 7, 14 and 21\u00a0days after hydrogel and cell transplantation. In control experiments, cells were injected without hydrogel, in NaCl solution only. PCR analysis using human probes and abovementioned muscle groups revealed that in the acute muscle injury model human transcripts were detectable in all samples until day 7 after hydrogel and cell transplantation (Supplementary Table 5).<\/p>\n Evaluation of the structure and functionality of mouse dystrophic muscles transplanted with hydrogels alone or with myoblasts derived from induced pluripotent stem cells<\/p>\n In the next set of experiments, we transplanted hydrogels alone or with myoblasts derived from hiPSCs to dystrophic muscles of SCID\/mdx that serve as an animal model of Duchenne muscular dystrophy. Dystrophic muscles that were not treated served as a control; they are further described as intact muscles. The treadmill test performed 28\u00a0days after hydrogel transplantation alone or with cells (DMD or LMN\u03b21 myoblasts) did not reveal any significant differences between all muscle groups (Supplementary Fig.\u00a07A,B). After the treadmill test, the mice were sacrificed and weighted and then the muscles were also isolated and weighted (Supplementary Table 3). The body weight ranged between 24.54 and 31.51\u00a0g (Supplementary Table 3). No significant differences in body or muscle weight were found (Supplementary Fig.\u00a08).<\/p>\n To follow the fate of transplanted cells we performed a similar analysis as in the case of the acute injury model, i.e., performed a RT-qPCR analysis using probes for human GAPDH, HPRT, ACTB and dystrophic mouse muscles collected on day 0, 3, 7, 14, 21, and 28 after hydrogel and cell transplantation. In control experiments, cells were injected without hydrogel, in NaCl solution only. RT-qPCR analysis using human probes and above-mentioned muscle groups revealed that in the dystrophic muscle injury model human transcripts were detectable in all samples only on the day of the injection, and then only in single samples collected at other time points (Supplementary Fig.\u00a05). Despite the elimination of cells, we performed comprehensive analysis of all SCID\/mdx muscle groups collected at 28\u00a0days after introduction of hydrogel alone or with cells, as well as of intact muscles. Comparison of muscle structure, including CSA analysis, revealed that dystrophic muscles injected with hydrogels and DMD cells were characterized by fiber size distribution similar to intact muscles (Fig.\u00a05<\/a>A, Supplementary Fig.\u00a09A). In contrast, muscles injected with hydrogels and LMN\u03b21 cells or hydrogel alone had significantly more smallest fibers in comparison to intact muscles (Fig.\u00a05<\/a>B,C, Supplementary Fig.\u00a09B,C) or muscles injected with hydrogels and DMD cells (Fig.\u00a05<\/a>D,E, Supplementary Fig.\u00a010A,B). They were also characterized by lower number of the largest fibers (CSA above 3750\u00a0\u00b5m) than intact muscles (Fig.\u00a05<\/a>B,C, Supplementary Fig.\u00a09B,C). When comparing muscles injected with hydrogels alone with those injected with hydrogels and LMN\u03b21 cells, the latter had significantly more smallest fibers (Fig.\u00a05<\/a>F, Supplementary Fig.\u00a010C). This may indicate that dystrophic muscles injected with hydrogels and LMN\u03b21 cells are less mature than muscles transplanted with hydrogels which are less mature than muscles injected with hydrogels and DMD cells (Supplementary Table 6). The last group of muscles injected with R1 hydrogel and DMD cells had more smallest fibers than muscles transplanted with functionalized hydrogels and DMD cells as well as intact ones, which again may indicate that the latter are more mature (Supplementary Table 6). Furthermore, the number of regenerating fibers with centrally located nuclei was elevated in few muscle groups, i.e., injected with R1 or R4-IL4 hydrogels alone, as well as after transplantation of DMD cells with functionalized hydrogels, that is, R1-SDF1 or R4-IL4, and also in muscles injected with R4-IL4 hydrogel and LMN\u03b21 cells (Supplementary Fig.\u00a07C). Histological analysis of Masson\u2019s trichrome stained muscle sections indicated that the level of fibrosis was similar in all analyzed muscle groups, i.e., it was not enhanced by any hydrogel or hydrogel and cell transplantation (Supplementary Fig.\u00a07D).<\/p>\n Fig. 5 Muscle CSA analysis after transplantation of hydrogels modified with IL4 or SDF1 mimicking peptides or control ones, alone or with hiPSC-derived myoblasts into dystrophic muscles of SCID\/mdx mice. All analyses were performed 4\u00a0weeks after hydrogel transplantation. For each indicated group, at least samples from 3 animals were analyzed. Five photos of the fields of view were analyzed for each muscle (i.e. separate biological sample). Then, the values for each muscle were averaged and subjected to statistical analysis. Boxes indicate the fiber area category in which significant differences were found between muscle groups. Percentage of fibers with different CSA in SCID\/mdx muscles injected with: (A) hydrogels and DMD cells as well as control muscles; (B) hydrogels and LMN\u03b21 cells as well as control muscles; (C) hydrogels and control muscles; (D) hydrogels and LMN\u03b21 cells as well as hydrogels and DMD cells; (E) hydrogels as well as hydrogels and DMD cells; (F) hydrogels and LMN\u03b21 cells as well as hydrogels.<\/p>\n Analysis of myogenesis, angiogenesis, and neurogenesis markers revealed a significantly higher level of Myh2, Myh3, Myog, Pax7, Vwf, Cd31, Lrp4 and Achr expression in dystrophic muscles injected with the R1-SDF1 hydrogel (Fig.\u00a06<\/a>A\u2013C). The level of mRNAs encoding other genes analyzed (Myh7, Dmd, Vegfa, Flt1, Musk, Dok7, Rapsn) was comparable between these muscles and intact ones (data not shown). In the case of Lrp4, its significantly higher expression level was also found in dystrophic muscle injected with R1 hydrogel and DMD cells compared to all other samples, except the muscle transplanted with R1-SDF1 (Fig.\u00a06<\/a>C). No other significant enhancement was observed for all other genes analyzed in dystrophic muscles injected with hydrogels alone or with cells. Their expression level was similar to or lower than that in intact muscles. Thus, the obtained results indicate that, in contrast to the acute muscle injury model, co-injection of cells and hydrogels to dystrophic mouse muscles did not enhance the beneficial effect of hydrogel alone, probably due to the rapid disappearance of injected cells. The most profound effect in dystrophic muscles, i.e. enhanced expression of myogenesis, angiogenesis, and neurogenesis markers, was found in muscles transplanted with R1-SDF1 hydrogel. The preinjury of dystrophic muscles did not change the outcome of hydrogel transplantation, alone or with cells, including their, i.e. transplanted cell persistence in muscles after injection (data not shown).<\/p>\n Fig. 6 Expression of selected markers of myogenesis, angiogenesis and neurogenesis in SCID\/mdx muscles after transplantation of hydrogels modified with IL4 or SDF1 mimicking peptides alone or with hiPSC-derived myoblasts. (A) Myogenesis markers (Myh2, Myh3, Myog, Pax7); (B) Angiogenesis markers (Vwf, Cd31); (C) Neurogenesis markers (Lrp4, Achr). For (A\u2013C) mean RQ (2-\u0394\u0394CT) values with standard deviation of at least 3 separate biological samples (obtained from different animals with results presented as dots) are shown. Intact muscles served as a reference sample. *p\u2009\u2264\u20090.05, **p\u2009\u2264\u20090.01, ***p\u2009\u2264\u20090.001, ****p\u2009\u2264\u20090.0001.<\/p>\n","protected":false},"excerpt":{"rendered":"Muscle structure after transplantation of hydrogels modified with IL4 or SDF1 mimicking peptides In the initial experiments, muscles…\n","protected":false},"author":2,"featured_media":568870,"comment_status":"","ping_status":"","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[5],"tags":[114,4068,135856,85,257421,46,4069,141,122250,257422,232179],"class_list":["post-568869","post","type-post","status-publish","format-standard","has-post-thumbnail","category-business","tag-business","tag-humanities-and-social-sciences","tag-hydrogels","tag-il","tag-interleukin-4","tag-israel","tag-multidisciplinary","tag-science","tag-skeletal-muscle","tag-stromal-derived-factor-1","tag-transplantation"],"_links":{"self":[{"href":"https:\/\/www.newsbeep.com\/il\/wp-json\/wp\/v2\/posts\/568869","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.newsbeep.com\/il\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.newsbeep.com\/il\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.newsbeep.com\/il\/wp-json\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/www.newsbeep.com\/il\/wp-json\/wp\/v2\/comments?post=568869"}],"version-history":[{"count":0,"href":"https:\/\/www.newsbeep.com\/il\/wp-json\/wp\/v2\/posts\/568869\/revisions"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.newsbeep.com\/il\/wp-json\/wp\/v2\/media\/568870"}],"wp:attachment":[{"href":"https:\/\/www.newsbeep.com\/il\/wp-json\/wp\/v2\/media?parent=568869"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.newsbeep.com\/il\/wp-json\/wp\/v2\/categories?post=568869"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.newsbeep.com\/il\/wp-json\/wp\/v2\/tags?post=568869"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}![\"Fig.](\"https:\/\/www.newsbeep.com\/il\/wp-content\/uploads\/2026\/07\/41598_2026_49704_Fig2_HTML.png\")
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