Transplantation is essential and crucial for individuals suffering from end-stage organ failure diseases. However, there are still many challenges regarding these procedures, such as high rates of organ rejection, shortage of organ donors, and long waiting lines. Thus, investments and efforts to develop laboratory-grown organs have increased over the past years, and with the recent progress in regenerative medicine, growing organs in vitro might be a reality within the next decades. One of the many different strategies to address this issue relies on organoid technology, a miniaturized and simplified version of an organ. Here, we address recent progress on organoid research, focusing on transplantation of intestine, retina, kidney, liver, pancreas, brain, lung, and heart organoids. Also, we discuss the main outcomes after organoid transplantation, common challenges faced by these promising regenerative medicine approaches, and future perspectives on the field.

Non-syndromic cleft lip with or without cleft palate (NSCLP), the most common human craniofacial malformation, is a complex disorder given its genetic heterogeneity and multifactorial component revealed by genetic, epidemiological, and epigenetic findings. Epigenetic variations associated with NSCLP have been identified; however, functional investigation has been limited. Here, we combined a reanalysis of NSCLP methylome data with genetic analysis and used both in vitro and in vivo approaches to dissect the functional effects of epigenetic changes. We found a region in mir152 that is frequently hypomethylated in NSCLP cohorts (21-26%), leading to mir152 overexpression. mir152 overexpression in human neural crest cells led to downregulation of spliceosomal, ribosomal, and adherens junction genes. In vivo analysis using zebrafish embryos revealed that mir152 upregulation leads to craniofacial cartilage impairment. Also, we suggest that zebrafish embryonic hypoxia leads to mir152 upregulation combined with mir152 hypomethylation and also analogous palatal alterations. We therefore propose that mir152 hypomethylation, potentially induced by hypoxia in early development, is a novel and frequent predisposing factor to NSCLP.

Auriculocondylar Syndrome (ACS) is a rare genetic disease that affects structures from the first and second pharyngeal arch, resulting in micrognathia, auricular malformations and dysplasia of the mandibular condyle. The ACS mostly segregates in an autosomal dominant manner with incomplete penetrance, but there are few reported cases whose pattern is autosomal recessive.In addition, there is inter and intra-familial variable expressivity and also genetic heterogeneity.To date, variants have been identified in three genes involved in the EDN1-DLX5/6 pathway: GNAI3, PLCB4 and EDN1. Studies carried out in our laboratory suggested the existence of a fourth locus related to the syndrome, since mutations in the 3 genes already associated with ACS were excluded and linkage studies in a Brazilian family with 11 affected individuals indicated that the variant is located on chromosome 7 (7p21.1-p15.2). In a collaborative work with the University of Oxford, we identified a tandem duplication of circa 400kb in the candidate region of chromosome 7, which includes the HDAC9 gene, in the individuals of this Brazilian family affected by the ACS.The present study aimed, therefore, to investigate the causative variant of ACS in this family in order to verify whether the duplication identified on chromosome 7 has a causal relationship with the phenotype, since it is the only family with changes in this region so far. To answer our research question, we used induced pluripotent stem cells (iPSC) as a model to recapitulate different stages of embryonic development and to investigate which cellular processes and alterations in gene expression could be associated with ACS in these patients. We observed increased expression of the HDAC9 and TWIST1 genes and decreased migration in neural crest cells. We also observed a dysregulation in osteogenic and chondrogenic differentiation in affected cells. These findings support the hypothesis of pathogenicity of the duplication within HDAC9 and its involvement in ACS phenotype in this family.

12 13 Non-syndromic cleft lip/palate (NSCLP), the most common human craniofacial 14 malformations, is a complex disorder given its genetic heterogeneity and multifactorial 15 component revealed by genetic, epidemiological and epigenetic findings. Association of 16 epigenetic variations with NSCLP has been made, however still of little functional investigation. 17Here we combined a reanalysis of NSCLP methylome data with genetic analysis and used both 18 in vitro and in vivo approaches to dissect the functional effects of epigenetic changes. We found 19 a frequent differentially methylated region in mir152, hypomethylated in NSCLP cohorts (21-20 26%), leading to mir152 overexpression. In vivo analysis using zebrafish embryos revealed that 21 mir152 upregulation leads to craniofacial impairment analogue to palatal defects. Also, we 22 demonstrated that zebrafish embryonic hypoxia leads to mir152 upregulation combined with 23 mir152 hypomethylation and also analogue palatal alterations. We therefore suggest mir152 24 hypomethylation, potentially induced by hypoxia in early development, as a novel and frequent 25 predisposing factor to NSCLP. 26 molecular alteration for complex traits 42 . Here, we attempted to identify both group and 53 individual-specific methylation changes using previously published methylome data on NSCLP. 54We identified individual methylation changes in known NSCLP candidate regions and also 55 mir152 hypomethylation in 26% of our discovery cohort. This result was replicated in an 56 independent cohort and validated through functional in vitro and in vivo assays. Finally, we 57 demonstrated how hypoxia, a known environmental risk factor for NSCLP, can modulate such 58 changes. 59 60Results 61 62 mir152 is a frequent differentially methylated region in the Brazilian NSCLP cohort 63 64We conducted differential methylation analysis at the gene level using the whole 65Brazilian NSCLP 450K dataset (66 NSCLP vs 59 controls 24 , and looked for the top 5 DMRs 66 ranked by RnBeads, which combines adjusted p-value to methylation difference and 67 methylation quotient. Those top DMRs were, in order of ranking: top 1, an intronic region of 68 CROCC at 1p36.13; top 2, an intronic region of FAM49B at 8q24.21; top 3, an intronic region of 69 NLK at 17q11.2; top 4, a non-coding region comprising mir152 at 17q21.32; and top 5, an 70 exonic region of PRAC2 and comprising mir3185 also at 17q21.32 (Figure 1a; Supplementary 71 Table 2). Among those genes, mir152 (adjusted p-value= 8.20E-06, beta-difference = -0.04) 72 was the only with enriched expression during palatal embryogenesis in human and mouse, 73 according to Sysface (Systems tool for craniofacial expression-based gene discovery) online 74 tool. Moreover, mir152 has already been identified as a DMR during normal murine palatal 75 development 53 and suggested as a central regulator of downstream mRNAs encoding proteins 76 known to play pivotal roles in orofacial development 54 , however still with no clear evidence of 77 association with NSCLP. Concur...