extfieldsofstudy: Medicine Biology

text:
The promising novel biomarkers and candidate small molecule drugs in kidney renal clear cell carcinoma: Evidence from bioinformatics analysis of high‐throughput data

Abstract Background Kidney renal clear cell carcinoma (KIRC) is the most common subtype of renal tumor. However, the molecular mechanisms of KIRC pathogenesis remain little known. The purpose of our study was to identify potential key genes related to the occurrence and prognosis of KIRC, which could serve as novel diagnostic and prognostic biomarkers for KIRC. Methods Three gene expression profiles from gene expression omnibus database were integrated to identify differential expressed genes (DEGs) using limma package. Enrichment analysis and PPI construction for these DEGs were performed by bioinformatics tools. We used Gene Expression Profiling Interactive Analysis (GEPIA) database to further analyze the expression and prognostic values of hub genes. The GEPIA database was used to further validate the bioinformatics results. The Connectivity Map was used to identify candidate small molecules that could reverse the gene expression of KIRC. Results A total of 503 DEGs were obtained. The PPI network with 417 nodes and 1912 interactions was constructed. Go and KEGG pathway analysis revealed that these DEGs were most significantly enriched in excretion and valine, leucine, and isoleucine degradation, respectively. Six DEGs with high degree of connectivity (ACAA1, ACADSB, ALDH6A1, AUH, HADH, and PCCA) were selected as hub genes, which significantly associated with worse survival of patients. Finally, we identified the top 20 most significant small molecules and pipemidic acid was the most promising small molecule to reverse the KIRC gene expression. Conclusions This study first uncovered six key genes in KIRC which contributed to improving our understanding of the molecular mechanisms of KIRC pathogenesis. ACAA1, ACADSB, ALDH6A1, AUH, HADH, and PCCA could serve as the promising novel biomarkers for KIRC diagnosis, prognosis, and treatment.

| INTRODUCTION
Renal cell carcinoma (RCC), also known as renal cell adenocarcinoma, accounts for more than 90% of primary renal neoplasms and pelvic cancer and ranks among the top 10 malignant tumors in both men and women. The incidence and mortality of RCC have been increasing over the past two decades. (Bray et al., 2018;Escudier et al., 2016;Hsieh, Le, Cao, Cheng, & Creighton, 2018;Srigley et al., 2013) Kidney renal clear cell carcinoma (KIRC) is the most common subtype of RCC (70%-75%) and represents one of the most malignant genitourinary cancers. Nearly a quarter of patients were diagnosed with distal metastasis and advanced regional disease. Although a major breakthrough has been made in the molecular mechanisms and therapeutic strategies for KIRC, the long-term overall survival of patients has not been significantly improved, especially in patients with metastatic disease, with 5-year survival <32%. (Linehan, 2012;Shuch et al., 2015) Therefore, identifying the promising novel diagnostic and prognostic biomarkers is urgently demanded, which will contribute to enhance our understanding of KIRC initiation and progression and promote early detection of KIRC. A previous study showed that Polybromo-1 plays an important role in the proliferation and metastasis of KIRC by regulating multiple metabolic pathways including PI3K signaling pathway, glucose uptake pathway, and hypoxia response genes. (Chowdhury et al., 2016) In the past decade, with the rapid development of microarray and RNA-sequencing technology, more and more biomarkers of tumor initiation, progression, and prognosis have been identified using bioinformatics analysis. (Sun et al., 2019;Yan et al., 2019) In this study, we integrated three microarray datasets from gene expression omnibus (GEO) database (GSE781, GSE6344, and GSE100666) to identify the differentially expressed genes (DEGs) between KIRC and adjacent normal tissues, aiming to explore and determine the promising novel biomarkers associated with pathogenesis and prognosis of KIRC. Meanwhile, we revealed some candidate small molecule drugs that could reverse the gene expression of KIRC based on the CMap database. The present study uncovered six novel biomarkers that were of great value in the diagnosis and prognosis of KIRC patients, which may contribute in revealing the molecular mechanisms of KIRC occurrence and progression. Figure 1 showed the workflow of our study.

| Data resources
We conducted a systematic search of GEO database for relevant microarray datasets in order to investigate the differential gene expression between KIRC and normal samples. GEO database (http://www.ncbi.nlm.nih.gov/geo/) is widely used to archive high-throughput microarray experimental data, original series, and platform records. In total, the gene expression profiles of GSE781 (Lenburg et al., 2003), GSE6344 (Gumz et al., 2007;Tun et al., 2010), and GSE100666 were downloaded from GEO database. These RNA profiles were provided on GPL96 (Affymetrix Human Genome U133A Array) and platforms GPL16951 (Phalanx Human OneArray Ver. 6 Release 1).

| Identification of DEGs
The original CEL files were downloaded and classified as KIRC and normal groups. The affy package of Bioconductor (http://www.bioconductor.org/) was used for data standardization and transforming raw data into expression values. The significance analysis of the empirical Bayes methods within limma package was applied to identify DEGs between KIRC samples and normal tissue sample. (Ritchie et al., 2015) The genes with the following cutoff criteria were considered as the significant DEGs: p < 0.05 and |logFC|>1.

| Functional enrichment analysis
We performed gene ontology (GO) enrichment analysis for these overlap DEGs. GO enrichment analysis is a common useful method to analyze biological process (BP), molecular functions (MF), and cellular components (CC) of genes

| Protein-protein interaction (PPI) network construction and module analysis
The Search Tool for the Retrieval of Interacting Genes database (STRING, https://string-db.org/) was used to construct the PPI network. (Szklarczyk et al., 2015) The interactions with a combined score >0.4 were considered significant. The PPI network was visualized through the Cytoscape software. (Smoot, Ono, Ruscheinski, Wang, & Ideker, 2011) Subsequently, Molecular Complex Detection (MCODE) plug-in of the Cytoscape software was applied to screen significant modules from the PPI network. The degree cutoff = 10, node score cutoff = 0.2, k-core = 2, and max. depth = 100 were used as selection criterion. (Bandettini et al., 2012) We also performed Go and KEGG pathway enrichment analysis for the most significant modules. In addition, the Networks Gene Oncology tool (BiNGO) plugin in Cytoscape was used to perform and visualize the biological process analysis of the hub genes. (Maere, Heymans, & Kuiper, 2005)

| Analysis and validation of hub genes
The hierarchical clustering of module genes was constructed by UCSC Cancer Genomics Browser (http://genome-cancer.ucsc.edu). A network of hub genes and their coexpression genes was established by cBioPortal online platform (http://www.cbioportal.org). The Gene Expression Profiling Interactive Analysis (GEPIA) was used to further confirm the reliability of the expression level of hub genes and their prognostic value. (Cerami et al., 2012;Gao et al., 2013;Tang et al., 2017) The hazard ratio (HR) with 95% confidence intervals (CI) of overall survival was calculated. The Kaplan-Meier curve and boxplot were visualized to present these relationships between gene expression and patients' prognosis. The human protein atlas (HPA, www.proteinatlas. org) database was used to determine the protein expression of the hub genes between KIRC and normal tissues based on immunohistochemistry.

| Identification of small molecules
We compared the overlap DEGs with those in the Connectivity Map database (CMap, http://www.broadinstitute.org/cmap/) to predict potential small active molecular that could reverse the current biological status of KIRC. (Lamb et al., 2006) First, the overlap DEGs were divided into upregulated and downregulated groups. Then different expression significant probesets from each group were selected for gene set enrichment analysis. Finally, the enrichment scores ranging from −1 to +1 were calculated. A positive connectivity value (closer to +1) indicated that the corresponding small molecules could induce the gene expression of KIRC, whereas a negative connectivity value (closer to −1) indicated greater similarity among the genes and the small molecules that could reverse the KIRC cell status.

| Identification of DEGs in KIRC
After the standardization of GSE781, GSE6344, and GSE100666 datasets, a total of 503 overlap DEGs expressed in KIRC tissues were identified with the Limma package. The volcano plot of DEGs of KIRC in each dataset was presented in Figure 2A. The Venn diagrams showed the 503 overlap DEGs among the three datasets (Figure 2Ba), consisting of 231 upregulated genes ( Figure 2Bb) and 250 downregulated genes (Figure 2Bc).

| Enrichment analyses
To better understand the biological functions of overlap DEGs among the three datasets, we performed GO function and KEGG pathway enrichment analysis using DAVID. For biological processes, GO analysis results indicated that upregulated and downregulated DEGs were significantly enriched in excretion, defense response, response to wounding, immune response, and regulation of response to external   IL10RA   LILRB2   CCR5   LCK  IL7  ACACB  ACSM3  PCCB  GCDH  FN1  C1QA  C1QB  TYROBP  CXCR4  ECHS1  SDC1  CTSS  TLR3  CD40  BCL6  HADH  ACAT1  ALDH4A1  ALDH6A1  ACAA1  AUH  homodimerization activity, glycosaminoglycan binding, and carbohydrate binding. Additionally, the results of KEGG pathway analysis revealed that these DEGs were mainly enriched in valine, leucine, and isoleucine degradation, propanoate metabolism, complement and coagulation cascades, butanoate metabolism, and cell adhesion molecules (CAMs). (Figure 3; Table 1).

| PPI network construction and module analysis
The PPI network among the DEGs was established by using the STRING database, with 417 nodes and 1912 interactions. (Figure 4a) Figure 4b presented the most significant module extracted from this PPI network by MCODE. Using KEGG pathway enrichment analysis, the genes in this module were mainly enriched in valine, leucine and isoleucine degradation, propanoate metabolism, and fatty acid degradation. (Table 2) Hierarchical clustering indicated that the module genes could basically differentiate the KIRC tissues from the noncancerous tissues. (Figure 5a) The biological process analysis indicated that the module genes were significantly related to cell cycle arrest, negative regulation of cell cycle, and cell cycle arrest in response to pheromone. (Figure 5b) ACAA1, ACADSB, ALDH6A1, AUH, HADH, and PCCA with high degree of connectivity were selected as hub genes.
The expression level of ACAA1, ACADSB, ALDH6A1, AUH, HADH, and PCCA in KIRC tissues was significantly downregulated among each dataset. (Figure 6). Table S1 showed the full names and function roles of these hub genes.

| Analysis and validation of hub genes
The hub gene expression and their prognostic correlation were further analyzed by GEPIA database. The mining of GEPIA database further confirmed that these hub genes were significantly downregulated in KIRC tissues compared to normal tissues. (Figure 7a) Based on the prognostic analysis of 516 KIRC patients in GEPIA database, we found that downregulation of ACAA1, ACADSB, ALDH6A1, AUH, HADH, and PCCA were correlated with significantly worse overall survival of patients. (Figure 7b) The expression level of these hub genes could represent the important prognostic biomarkers for predicting the survival of KIRC patients. Due to the gene expression not always consistent with its protein content, (Maier, Guell, & Serrano, 2009) we used HPA database to further analyze the protein level of hub genes. The immunohistochemical staining results indicated significantly higher positivity for ACAA1, ACADSB, ALDH6A1, AUH, HADH, and PCCA in adjacent normal tissues than in cancer tissues. (Figure 8) A network of the module genes and their coexpression genes was constructed using cBioPortal online platform. (Figure  9a) To further explore the molecular mechanism of the hub genes in KIRC, we predicted potential transcription factors and constructed a regulatory network of lncRNA, miRNA, and mRNA which could be involved in the expression of hub genes by GGBI analysis.(Gene-Cloud Biotechnology Information, Figure S1 & S2).

| Identification of related active small molecules
In order to screen and identify potential therapeutic drugs for KIRC, we uploaded these DEGs which were divided into upregulated and downregulated groups into the CMap database and then matched them with small molecule treatment. The top 20 most significant small molecules with their enrichment scores were shown in Table  3 and Figure 9b. The small molecules of pipemidic acid (enrichment score = −0.853) and dicloxacillin (enrichment score = −0.819) were related to highly significant negative scores, which could imitate the status of normal cells. These potential small molecule drugs could reverse the gene expression induced by KIRC, thus providing new directions and molecular mechanisms for the future development of new targeted drugs for treating KIRC. However, further studies were still required to confirm the value of these candidate small molecules in treating KIRC.

| DISCUSSION
KIRC is the most lethal urological disease and the incidence has increased over the past few decades. KIRC is also a heterogeneous histological disease and the mechanisms underlying its occurrence and progression are complex. (Van Poppel et al., 2011;Xue et al., 2012) Therefore, identifying the key genes related to the pathogenesis and prognosis of KIRC is urgently demanded. In the present study, we integrated gene expression profiles of GSE781, GSE6344, and GSE100666 from GEO database to screen the differentially expressed genes between KIRC and adjacent normal tissues by bioinformatics techniques. The purpose of our study was to find the novel diagnostic and prognostic biomarkers for KIRC. Additionally, we also identified the candidate small molecule drugs that could reverse the tumoral status of KIRC in order to provide new directions for the future development of new drugs for treating KIRC. A total of 503 overlap DEGs were found among the three datasets, including 231 significantly upregulated genes and 250 downregulated genes. We analyzed the biological process, molecular function, and cellular component of these overlap DEGs using GO enrichment analysis in order to explore their role in the occurrence and development of KIRC. Excretion, defense response, and response to wounding were F I G U R E 8 Representative immunohistochemistry staining results reveal the protein level expression of hub genes in KIRC and normal tissues ALDH6A1 Control ACADSB ACAA1 PCCA HADH AUH KIRC Control KIRC the top three significantly major functions among the biological process. Molecular function enriched for the DEGs were mainly within identical protein binding, protein dimerization activity, and protein homodimerization activity. Changes in cell component were mainly associated with plasma membrane part, integral to plasma membrane, and intrinsic to plasma membrane. Furthermore, the results of KEGG pathway suggested these DEGs were significantly enriched in valine, leucine and isoleucine degradation, propanoate metabolism, complement and coagulation cascades, butanoate metabolism, and cell adhesion molecules. The cell adhesion molecules are widely involved in the intercellular and cellular-extracellular matrix interactions of tumors. Some adhesion molecules play an important role in tumor recurrence, metastasis, and invasion. (Okegawa, Pong, Li, & Hsieh, 2004) Based on the construction of PPI network and module analysis, ACAA1, ACADSB, ALDH6A1, AUH, HADH, and PCCA with high degree of connectivity were selected as hub genes. They were significantly downregulated in KIRC tissues compared with normal tissues. We used GEPIA database to further validate the results of bioinformatics analysis. Similarly, the mining of GEPIA database also confirmed that these hub genes exhibited significant differences in expression between KIRC and normal tissues. More importantly, these hub genes have important value in predicting the survival of KIRC patients. The low expression level of ACAA1, ACADSB, ALDH6A1, AUH, HADH, and PCCA was significantly correlated with the prognosis of patients. The immunohistochemical staining results revealed that the expression level of hub gene was consistent with their protein expression level, which further validated the accuracy of our findings. The present study first revealed six key genes with the diagnostic, prognostic, and therapeutic value in KIRC. No studies have reported the role of these six key genes in the initiation and progression of KIRC. In order to further enhance the understanding of these hub genes in the pathogenesis of KIRC, we predicted the corresponding potential transcription factors and establish a regulatory network of lncRNA-miRNA-mRNA for each hub gene. The construction of these regulatory networks will contribute in revealing the potential mechanism of these hub genes in KIRC initiation and progression. CENPK, a member of the centromeric protein family, was markedly upregulated in ovarian cancer tissues and associated with poor prognosis in patients. ALDH6A1 and ALDH6A1 were significantly upregulated in metastatic prostate cancer and could be used to predict the survival of patients. (Cho et al., 2018) A previous study showed that HADH knockdown can promote tumor cell migration and invasion by activing Akt signaling pathway. (Shen et al., 2017) Evidence indicated that ACADSB was also markedly downregulated in HCC cell lines. (Nwosu et al., 2018) However, no studies have reported the effects of hub genes on the initiation and progression of KIRC. The above studies indicated these hub genes could play an important role in the occurrence and development of KIRC.
In addition, we used the CMap database to determine a set of potential small molecules which may imitate the gene expression of normal cells. The small molecules with higher negative enrichment scores meant that they are more likely to reverse KIRC gene expression. This work will contribute to developing new target therapeutic drugs for KIRC. The efficacy and safety of pipemidic acid, the most significant small molecule (enrichment score = −0.853), has not been investigated as yet in KIRC. Meanwhile, the relationship between dicloxacillin (enrichment score = −0.819) and KIRC remains little known. Therefore, further studies were urgently demanded to reveal the huge potential of these listed small molecules in treating KIRC.
In conclusion, through a comprehensive bioinformatics analysis for the microarray datasets of KIRC, we first uncovered six novel key genes associated with KIRC initiation and progression. These hub genes could serve as promising novel biomarkers for the diagnosis, prognosis, and treatment of KIRC. A series of detailed analysis were further confirmed their vital roles on the pathogenesis of KIRC. In addition, this study identified a group of candidate small molecule drugs, which could provide new directions for the development of new targeted drugs for KIRC. Taken together, this work provided new insights for KIRC to integrate multiple biomarkers in clinical practices.

CONFLICTS OF INTERESTS
None.


extfieldsofstudy: Biology Medicine

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Past, Present, and Future Strategies for Enhanced Assessment of Embryo's Genome and Reproductive Competence in Women of Advanced Reproductive Age

Recent advancements in genomic analysis allow testing of an increasing number of genetic features in human preimplantation embryos. Typical single gene mutation and whole chromosomes testing can now be integrated with assessment of mitochondrial DNA and polygenic conditions. Diagnostic expansion into epigenetic and transcriptomic assessment in the near future are potential technological targets which may improve the prognostic outlook of patients of advanced reproductive age and overall in vitro fertilization (IVF) treatment outcomes. In this review, we discuss the technological progress of recent years and their future applications in preimplantation genetic testing in IVF.

INTRODUCTION
Preimplantation genetic testing (PGT) is a methodology designed to assess the genetic complement of embryos generated during in vitro fertilization (IVF) treatments. Its current applications include both the detection of monogenic disorders and chromosome copy number. Over the years the molecular strategies employed for PGT have changed and improved, taking advantage of the technological progressions introduced in molecular genetics (Figure 1, Table 1). In parallel, improvements in culture systems and cryopreservation protocols employed in IVF allowed the production of more robust results from genetic testing and additional analytical flexibility and opportunity, enabling simultaneous evaluation of reproductive features of IVF embryos beyond genetic and chromosomal status (Figure 1, Table 1).
In this article, we review the molecular and embryological strategies employed so far, including future technological developments for increasing the scope of genetic assessment and complementary non-invasive approaches especially focused on counteracting the genetic risks associated with advanced reproductive age. FIGURE 1 | Timeline of introduction to clinical practice of embryological and analytical achievements. Analytical strategy, dark gray bars section: FISH was first employed for assessment of a limited amount of chromosomes in mid '90s, followed by microarray-based comprehensive chromosomal screening techniques (e.g., aSNP, aCGH) in mid 2000's. Comprehensive quantitative PCR methods were introduced in '10s, shortly followed by NGS-based methods, which is now employed for most of chromosomal screening analyses, progressively replacing other less sensitive, more expensive and labor intensive techniques. Embryo culture strategy, light gray bars section: Blastocyst culture is still being introduced into clinical settings (ongoing since 2000's). Similarly, embryo vitrification is increasingly being adopted since its introduction in mid 2000's. Trophectoderm biopsy is currently the gold standard for embryo genetic assessment; however, it may become replaced by non-invasive approaches for certain applications (i.e., PGT-A) should they demonstrate high sensitivity and specificity in large clinical trials. Diagnostic/prognostic coverage, colored bars section: Cleavage stage biopsy: FISH was first employed in cleavage stage embryos. It was later completely replaced by comprehensive CCS strategies, which enabled analysis of all chromosomes. Cleavage stage biopsy is still employed by IVF laboratories not equipped for blastocyst culture. Blastocyst stage biopsy: Trophectoderm cells provide the most robust specimen to generate genetic analysis on. Therefore, this strategy is applicable to all diagnostic approaches including embryo karyotyping, expanded mutation screening, mitochondrial DNA analysis and mosaicism detection, as well as future additional clinically employable methodologies analyzing epigenetic and trascriptomic features of the embryo. Non-invasive assessment: Genetic analysis of spent culture media provides the safest and least invasive approach. Clinical application of this strategy would allow assessment of the chromosomal complement of embryonic cells as well as the analysis of embryo-derived transcripts with paracrine/autocrine effect (i.e., miRNA). Its suitability for analysis of factors with typical intracellular localization (i.e., mitochondria, epigenetic features) is still being investigated. It is unlikely that niPGT will be able to provide meaningful data on mosaicism status of the embryo. Nonetheless, this application requires further data to reach an exhaustive answer.

BIOPSY STRATEGY
Embryo biopsy represents a crucial step in embryo genetic assessment both from biological and technical standpoints. Originally, embryo biopsy was performed on cleavage stage embryos by removing one (or two) cell(s) from an 8-cell embryo. Over the years, once enough data was collected, this practice has shown several limitations (1). Most importantly, the removal of one cell from cleavage stage embryo was shown to have an impact on its developmental and reproductive potential due to the risk of removing cells already committed to inner cell mass lineage differentiation (2). The series of molecular events ultimately leading to symmetry breaking and the timing and progression of cell lineage commitment are poorly characterized in human embryos. Additionally, the homogeneity in appearance of cleavage stage blastomeres hinders any possibility to evaluate embryo's cell lineage commitment progression morphologically, thus leaving to chance the risk of removing an ICM-committed cell during biopsy.
The development of extended culture systems has allowed the postponement of embryo biopsy to the blastocyst stage, where embryo genome activation is complete and cell lineage commitment becomes morphologically detectable. Additionally, by collecting 5-8 cells from the trophectoderm (TE) wall, the proportion of the embryonic biomass removed at the blastocyst stage is lower compared to cleavage stage (3-5 vs. 12%, respectively) and selectively obtained from the extra-embryonic lineage. The intrinsic advantages of blastocyst biopsy, combined with the development of comprehensive chromosome screening platforms (CCS), led to the development of a more robust diagnostic strategy. Firstly, the higher number of cells collected provides a larger amount of DNA template for downstream amplification and analysis. This feature reduces significantly the proportion of failed amplification and allele drop out events, increasing technical reproducibility and diagnostic rates. Secondly, multiple cell analysis generates more robust results compared to single cell analysis, minimizing the risk of false positive and false negative results associated with single cell analysis. Lastly, TE biopsy followed by vitrification yields to higher survival rates compared to slow freezing of biopsied cleavage stage embryos (3). The higher results are due to the lower water content of TE cells compared to blastomeres, the reduced risk of intracellular ice crystal formation associated with vitrification and the higher structural flexibility of blastocysts compared to cleavage stage embryos (4). All these factors have contributed to the development of a global strategy for the investigation of embryos genetic features, based on multicellular samples supplying reliable source of template DNA for robust downstream analysis. Finally, from a clinical viewpoint, the exclusive analysis of developmentally competent embryos resulted in a more costeffective diagnostic strategy compared to previous approaches employing PBs or blastomeres, leading to a rapid expansion of PGT practice to more clinics and patients over the last years. Indeed, a recent survey from the ESHRE PGD consortium showed an increasing trend toward blastocyst biopsy in recent years and that in 2016 it was the main strategy for preimplantation genetic testing of embryos (roughly 60% of all PGT procedures, data from ESHRE PGD consortium at the 2018 Annual Meeting in Barcelona).
Patients in advanced reproductive age opting for PGT analysis benefit from blastocyst biopsy strategy in three main ways. Through extended culture they (a) allow the assessment of their embryos' ability to develop passed the cleavage stage (Day 3), (b) reduce extra analytical costs by limiting the number of embryos to test, and (c) receive robust diagnostic results upon which meaningful clinical decisions can be made.

CHROMOSOMES
Chromosome analysis has been employed clinically since the mid '90s (5,6). This type of analysis is performed to ensure that the embryo transferred to the patient has a correct number of chromosomes, thus reducing the risks of abnormal pregnancy, miscarriage, and failed implantation. Initially, this diagnostic strategy was performed by fluorescent in situ hybridization (FISH), involving the spreading of a single cell biopsied from a cleavage stage embryo on a glass slide and the hybridization of its DNA with chromosomespecific fluorescent probes. Several limitations of this approach have been described in the literature, including the limited amount of chromosomes that could be simultaneously assessed and the high incidence of false positive results (7,8). The limited number of chromosome probes evaluated also meant that some aneuploidies were left untested, resulting in undetected aneuploid embryos being transferred. In the mid 2000's, it became clear that these shortcomings and diagnostic unreliability, coupled with the negative consequences of cleavage stage biopsy, were compromising clinical outcomes of patients undergoing PGT, highlighting the necessity of a safer, more robust and precise strategy (9). Subsequently, the development of comprehensive chromosome screening technologies (CCS), including comparative genomic hybridization arrays (aCGH) and single nucleotide polymorphisms arrays (SNP arrays) and quantitative polymerase chain reaction (qPCR), provided significant improvement to PGT clinical application. These technologies not only are able to accurately evaluate all 24 chromosomes in a single analysis, but also are applicable to single cells with sufficient accuracy. When tested on single cell from fibroblast cell lines with known karyotype, all platforms provided accuracy rates above 98% for whole chromosome aneuploidies (10)(11)(12)(13)(14). The largest comparative study between two methodologies (aCGH and qPCR) conducted on embryo biopsies reported high concordance across the two platforms (15). In this study, qPCR and aCGH showed similar sensitivity (98.2 vs. 98.8%, respectively, not significant), whereas qPCR displayed a significantly higher specificity compared with aCGH (99.9 vs. 99.6%, respectively, P = 0.01) (15). Despite the need for larger comparative studies, technological performance appears to be similar across all platforms when standard criteria for diagnosis of whole chromosome aneuploidies are used.
All CCS strategies allow parallel sample analysis and produce higher throughput compared to FISH. Also, some of the CCS strategies now available avoid time consuming and high labor-intensive steps required for FISH analysis, allowing more reproducible and streamlined processing conditions (i.e., qPCR, aCGH). In recent years, Next Generation Sequencing (NGS) platforms have been adapted for embryo aneuploidy testing using low-depth genome sequencing and copy number variation analysis. Due to its sensitivity, coupled with further extended chromosome coverage, NGS provides higher accuracy in the assessment of sub-chromosomal abnormalities (i.e., segmental aneuploidies) compared to previous CCS methods (16). Additionally, NGS is currently employed for the detection of chromosomal mosaicism, where two karyotypically different cell populations coexist in the same embryo. Despite lacking significant level of diagnostic validation, NGS was suggested to be able to detect low-level mosaicism (i.e., 20%) and accurately discriminate the proportion of cells showing abnormal karyotype (17). Nonetheless, mosaicism detection at low and high levels (e.g., 20 and 80%, respectively) is yet to be confirmed as a true biological finding, rather than a technical variation, hence its clinical impact still requires evidence support (18)(19)(20). This point is of extreme importance, especially for patients producing a limited amount of embryos per cycle, like advanced maternal age patients. Indeed, no embryos should be wasted due to diagnostic uncertainties, even more so in those cases where patients are unlikely to generate more embryos. Today, the main advantage provided by NGS in PGT is the possibility to analyze multiple samples in parallel, thus bringing a significant reduction of both costs and sample running time and allowing a wider accessibility to PGT for patients worldwide. Similarly to other qPCR platforms, NGS is compatible with combined assessment of both aneuploidy and single gene mutation, where the initial round of whole genome amplification (WGA) is integrated with targeted amplification of loci of interest (21)(22)(23).
The latest technology appearing in the preimplantation genetics landscape is Nanopore sequencing. This sequencing strategy is completely different from NGS as it relies on voltage changes occurring in protein nanopores fixed across a flow-cell membrane. Pre-amplified single-stranded DNA molecules are driven through the nanopores by an electrical current, whilst variable resistivity produced by the passage of each nucleic base is recorded (24). This information is then translated into DNA sequences that are subsequently aligned to a reference DNA and analyzed for chromosomal copy number variation. A small cohort study has recently shown that this technology is able to identify whole chromosome abnormalities in blastocyst biopsies with a sensitivity equal to NGS (25). Nanopore technology presents several advantages including a significantly lower price per analytical unit (∼$1,000 for a MinION vs. >$99,000 for a MiSeq), a smaller footprint in the laboratory, and the possibility of in-house genetic analysis for IVF laboratories, thus reducing overall time for analysis (∼2 h) and allowing for fresh embryo transfer after PGT. On the other hand, Nanopore strategy doesn't allow detection of mosaicism or segmental aneuploidies unless deeper sequencing experiments are performed. Additionally, Nanopore single-base calling sensitivity and precision requires validation to determine its suitability for more hi-depth sequencing tasks. Moreover, its implementation in IVF laboratories would still require technical expertise, dedicated clean room facilities for pre-amplification steps and WGA, as well as professional accreditation to carry out molecular biology diagnostics.
Current technology allows PGT laboratories to handle high volumes of samples for CCS tests, providing a very valuable tool for couples of advanced reproductive age undergoing IVF. IVF patients of advanced maternal age mainly benefit from PGT by avoiding the transfer of chromosomally impaired embryos. In these patients, the likelihood of producing abnormal embryos is significantly higher than in younger populations, hence aneuploidy testing is often recommended. Although preimplantation genetic testing for aneuploidies (PGT-A) doesn't improve cumulative clinical pregnancy rates per cycle started (as it can't improve or repair chromosomal abnormalities), it reduces the chance of miscarriage and failed implantation, thus increasing overall pregnancy rates per transfer and minimizing time to pregnancy (26,27).
Nonetheless, some controversies still surround the application of PGT-A, especially in relation to the clinical impact of mosaicism detection and segmental aneuploidies. Several groups are currently investigating these topics in an effort to clarify the real incidence of mosaicism in human embryos, whilst identifying a clinical management strategy that minimizes both embryo wastage and risks to patients.
Additionally, the clinical implementation of biopsy procedures can be challenging for smaller clinics both from a financial and logistic standpoint due to the purchase of equipment required for PGT (i.e., laser) and operators training.

SINGLE GENE MUTATIONS AND EXPANDED PREIMPLANTATION GENETIC TESTING
Prior to aneuploidy testing, preimplantation genetic testing was mainly employed for the detection of inheritable genetic conditions in embryos generated by couples with increased reproductive risk (28). This risk is determined by the presence of a pathological mutation in the genome of one or both the prospective parents. The main diagnostic strategy employed for single gene mutation detection has been only slightly changed from the first cases. Preimplantation genetic testing for mutations (PGT-M) involves the direct analysis of the region of the mutation performed by targeted amplification of the small region of the genome including the mutation site, followed by genetic variant detection by mini-sequencing or qPCR based genotyping. This analysis is strengthened by parallel multiplex amplification of informative highly polymorphic markers flanking the mutation site in order to counterbalance the risk of allele drop out at the mutation site and minimize the chance of no diagnosis. Similar to genetic testing for aneuploidy, the limiting factor in single gene disorder assessment is the amount of DNA available in single diploid cells, as a successful diagnosis is dependent on the detection of both alleles (maternal and paternal), each present in a single copy. Blastocyst biopsy is also beneficial in this type of analysis, increasing the starting template DNA material and thus reducing the risk of either DNA amplification failure (AF), target DNA contamination and allele dropout (ADO), in which one of the two alleles is preferentially amplified to the detriment of the other.
Karyomapping is a more recent diagnostic strategy for PGT-M that takes advantage of the presence of almost 300,000 SNPs scattered throughout the genome, which provide linkage analysis capabilities for virtually every region of interest. Using the data previously generated from both parents and an affected family member DNA samples, it is possible to reconstruct the segregation patterns of the mutation and assess its presence in the embryo. This indirect linkage analysis allows the detection of most pathological variants using a global platform that doesn't require patient-specific custom set-up. Despite its broad applicability, Karyomapping shows diagnostic limitations in cases where insufficient informative SNP markers are found in the region of interest (e.g., telomeric genes). Furthermore, the application of Karyomapping requires the analysis of at least one affected family member in addition to the two parents during set-up. If this reference sample is unavailable (around 30-40% of cases), a classic approach must be employed. Also, Karyomapping cannot be applied in cases involving de novo mutations, or when there are cases of recombination that make family samples noninformative (29), falling short of providing a universal approach for PGT-M analysis.
More recently, the development of NGS platforms has provided the possibility to combine the assessment of single gene mutations and aneuploidies on the same biopsy. Additionally, an alternative NGS-based approach aimed at the amplification of customizable genomic loci has been investigated (t-NGS) (21). This methodology involves a multiplex PCR that amplifies sequences necessary to assess both aneuploidy status and pathological mutations of a gene of interest, without providing data on the entire genome (21,30,31). However, due to the high sequencing depth required for accurate single nucleotide call in single cell genomics, NGS-based methods for PGT-M are still far from being cost-effective compared to previously established procedures and are therefore not commonly employed in current clinical practice. In the future, a reduction of sequencing costs, combined with improved automation, is expected, likely making NGS-based PGT-M feasible in a clinical context. Nonetheless, by using combined approaches, it is possible to assess both the presence of single gene disorders and karyotype of the embryo from the same biopsy specimen (23,32). This approach is especially recommended for patients of advanced maternal age carrying an inheritable genetic condition. In these cases, the embryo is at high risk not only of inheriting the genetic mutation, but also a chromosomal abnormality derived from age-related defective meiosis. Consequently, the cohort from whom the embryo is selected for transfer is dramatically reduced. For this reason, the treating clinician should carefully estimate patients' ovarian reserve and overall reproductive potential in order to provide them with adequate counsel.
The application of genome-wide technologies to PGT is also paving the way for screening of multifactorial conditions, where the assessment of multiple genes and epigenetic factors will enable clinical risk calculations for the onset of diseases not following Mendelian inheritance. This further evaluation of embryo's polygenic and multifactorial conditions might represent a possible solution for IVF patients, which may enable additional parameters for embryo ranking based on lower risk factors. Genome-wide SNP-array and NGS technologies have the potential to be employed clinically for the detection of relevant alleles included in polygenic risk scores panels (33). This approach, called "extended preimplantation genetic testing" (ePGT) can be employed to screen embryos for further genetic features, additional to known, full-penetrance conditions (autosomic dominant and recessive) running in the family (34). These include both incomplete penetrance conditions (i.e., cancer predisposition -BRCA1 and BRCA2 genes), but also multifactorial/polygenic conditions (i.e., congenital diabetes, cardiomyopathies, hypertension, hypothyroidism). This type of enhanced genetic assessment would allow a more thorough analysis of embryo's genetic inheritance, further restricting the total genetic risk passed on to the offspring, not only including mutations running in the family but also more common multifactorial conditions with strong genetic bases and de novo conditions and mutations exclusively present in the germinal line. With the evolution and full integration of genetics into clinical management of potentially every pathological and subpathological condition, expanded preimplantation genetic testing (ePGT) would enable the application of early interventions for medically actionable conditions in the prenatal or perinatal stages, providing tailored and efficient treatment of various conditions and improving overall prognosis and lifestyle of future generations.
Nonetheless, although providing additional testing capabilities and diagnostic coverage of pathological statuses, expanded genetic assessment would also involve potential incidental findings that may affect the life of the prospective parents, especially regarding late onset and incomplete penetrance conditions. Also, the actual prognostic predictive ability of several newly developed polygenic risk scores derived from population studies are yet to be validated and their potential clinical implementation should be further assessed and investigated. For this reason, the ethical consequences and the management of diagnostic results derived from expanded genetic testing of preimplantation embryos should be carefully evaluated for each individual case prior to its clinical implementation (35).
Ethical issues surrounding the application of ePGT and its preconceptional version ePCS (expanded preconception carrier screening, carried out on prospective parents rather than embryos) involve patient's well-being from a legal and psychosocial standpoint. Indeed, ePGT and ePCS should be offered to the general public only when its effects on voluntary informed consensus is granted by the patient. Additionally, the impact of ePGT/ePCS on individuals' psychological health and social fairness should be carefully investigated in order to warrant overall safety of the analysis. This assessment should include the impact of test results on psychological well-being, patient's perception of health and related false reassurance, as well as potential stigmatization, discrimination, legal inequity and unfairness derived from unfavorable results (36).
Although technical limitations and ethical concerns will require resolving prior to its clinical application, it is likely that ePGT will become applicable to the general IVF population in order to prevent the transmission of a wide spectrum of inheritable genetic conditions to their offspring.

NON-MENDELIAN GENETIC FACTORS
Parallel to nuclear genome analysis, additional genetic features are critical for both the transmission of inheritable conditions (i.e., mitochondrial disorders) and embryo developmental and implantation competence. Despite its ability to identify embryos with no reproductive potential (e.g., aneuploid embryos), PGT-A has limitations in identifying euploid embryos capable of implantation and further development. Indeed, only around 50% of euploid embryos transferred to patients go on to implant (37). Moreover, around 10% of the euploid embryos that implant end up miscarrying prior to 12 weeks of gestation, suggesting the need for more comprehensive testing approaches to assess extra molecular markers for reproductive competence. Additional embryo genetic features, including mitochondrial genome, epigenetic background and transcriptional activity could be analyzed to further assess their relation with embryo's reproductive capabilities.

Mitochondrial Diseases
Mitochondrial disorders are characterized by their exclusive maternal inheritance. The presence of pathological mutations in the mitochondrial genome (mtDNA) is a phenomenon due to both inheritance and spontaneous de novo occurrence due to the lack of DNA error checking mechanisms in the mitochondrion (which are instead present in the nucleus). Each mitochondrion contains multiple molecules of mtDNA, each of them potentially differing from the others (heteroplasmy). Each mitochondrion replicates autonomously within the cell, reaching a total number of hundreds of organelles. Mitochondrial pathological symptoms arise when the number of defective organelles passes the threshold of expression. Most of these pathological conditions involve energy production processes carried out in the mitochondrion, ultimately affecting several organs, especially targeting muscle and nervous tissues (i.e., myopathy, encephalopathy, neuropathy). For instance, mitochondrial disorders include Leber's hereditary optic neuropathy (LHON) where a point mutation occurs in one of the genes coding for protein subunits forming the NADH dehydrogenase enzyme, affecting the activity of oxidative phosphorylation complex I located in the mitochondrion inner membrane (nucleotide positions 11778 G to A, 3460 G to A and 14484 T to C, respectively in the ND4, ND1 and ND6 subunits) and eventually causing optic atrophy. A rare variant of this condition named LHON Plus also involves the loss of brain ability to control muscular activity including the cardiac muscle.
Due to the random nature of mitochondrial replication and consequent heteroplasmy ratio, mutated mtDNA load might require careful assessment prior to embryo transfer. Additionally, due to the high mutation rate in mtDNA and the complex penetrance mechanism, every person is at risk of harboring a potentially life-threatening mitochondrial condition.
For this reason, following technological and clinical validation, the integration of mitochondrial testing in conventional PGT would further expand the coverage of embryo genetic assessment, ensuring the identification of nuclear and mitochondrial mutation-free embryos. Similarly to cases where nuclear single gene mutations are investigated, aneuploidy assessment should also be performed for advanced maternal age. In order to produce realistic reproductive estimations, ovarian reserve should also be tested prior to commencing IVF/PGT-A/PGT-mitochondria treatment for these patients. Nonetheless, representativeness of biopsied cells of the mitochondrial constitution of the whole embryo still requires further investigation and extensive validation.

Mitochondrial Load as Embryo Viability Marker
The number of mtDNA molecules present in a biopsy has been proposed as a marker for embryo reproductive competence (38,39). It has been shown that significantly high concentration of mtDNA is associated with aneuploidy and lower implantation abilities. Concentration thresholds were proposed, however these parameters showed low sensitivity in defining embryo implantation potential (40). Additionally, confirmatory studies from other groups have failed to validate the initial findings and others have shown completely inversed outcomes (41,42). Nonetheless, the identification of molecular markers of embryo viability is of crucial importance for the field and, should initial findings be corroborated by additional evidence in future studies, mtDNA may be used for this purpose.

EPIGENETIC LANDSCAPE
Preimplantation embryos undergo major chromatin restructuring in the period between fertilization and implantation. Due to technical limitations associated with single cell genomics, these mechanisms and the extent of these processes have been ignored in humans until recent times. Animal models were first investigated, however, direct evidence of similar epigenetic strategies on humans have been lacking. Recent studies have shown the possibility of investigating chromatin rearrangements and gene accessibility in the early developmental stages following fertilization (43)(44)(45). Using single-cell chromatin overall omic-scale landscape sequencing (scCOOL-seq), Li et al. were able to simultaneously analyze several molecular epigenetic characteristics of embryonic genome including chromatin state, nucleosome positioning, DNA methylation and the interrelationship among different epigenetic layers in the same individual human preimplantation embryonic cells (44). This study provided a temporal landscape of the epigenetic status changes throughout development from gamete to blastocyst, revealing insights on DNA accessibility and transcription rates at different preimplantation stages. Another group investigating IVF embryos undergoing TE biopsy performed DNA sequencing after bisulfite conversion and used the methylation data to infer both transcription rates and chromosome copy number variation (46). According to this study, methylation levels of low-quality blastocysts diverge from those of high-quality blastocysts. Additionally, authors suggest that the presence of abnormally methylated regions is associated with the failure of embryonic development and live birth (46). This type of approach may be used in the future not only to assess embryo's chromosome composition but also to add prognostic outlook on its developmental and reproductive potential. Should oocyte aging be shown to impact epigenetic remodeling of maternal and embryonic genomes, the evaluation of these markers in embryo biopsies may potentially be used to further improve embryo selection between euploid embryos, especially in patients of advanced maternal age.

TRANSCRIPTOMICS ASSESSMENT
The impact of both infertility and IVF treatment on embryo development is poorly understood and although IVF is generally considered to be safe, it has been suggested that artificial conditions may have an impact on cell lineage differentiation processes and epigenetic conformation of embryo's genome that may result in fetal developmental and neonatal complications. Using an murine model, Giritharan and colleagues showed that ICM and TE of in vitro developed blastocysts showed lower transcriptional differences compared to in vivo developed blastocysts, suggesting a compromised cell differentiation mechanism (47). Additionally, a more recent study on human blastocysts showed that embryo transcriptional profiles are affected by the type of media used for culture, highlighting the impact of culture conditions on embryo development and the necessity of increase the understanding of this process (48). A recent study comparing RNA-Seq generated transcriptional maps of human blastocysts derived from young and older patients highlighted a significant age-dependent reduction in transcriptional activity for more than 800 genes (49). Biological functions of down-regulated genes included "cell cycle control" and "metaphase checkpoint regulation, " suggesting their concurrent role in the instauration of suboptimal chromosomal segregation processes in advanced maternal age patients. The translation of these results into a diagnostic approach improving reproductive outcomes for these patients is currently under evaluation.
Although these findings require further confirmation and validation, transcriptomic profiles may be targeted in future PGT assessment strategies to identify embryos with normal developmental competence and improve embryo selection.

NON-INVASIVE PGT
Currently, trophectoderm biopsy provides the most robust and reliable source of embryonic DNA for the analysis of embryo's genetic features. However, despite having been shown that TE biopsy doesn't affect embryo's reproductive potential (2), there is increasing interest in reducing or completely avoid intervention on the embryo for diagnostic purposes (50). Non-invasive assessment not only would completely avoid any potential injury to the embryo, but it would also predispose the genetic analysis to full automation and operator-independence. Approaches alternative to embryo biopsy that have been investigated thus far include both minimally invasive aspiration of the blastocoel fluid (BF) [e.g., Blastocentesis, (51-53)] and fully non-invasive analysis of spent culture media (54,55). Although initial tests of both these strategies have shown reduced overall diagnostic power (suboptimal DNA amplification rates, reduced concordance rate with TE results, inconsistence of results across groups) (56), the high potential benefits brought by their clinical application warrant further investigation. For example, the main setbacks from culture media testing have been reported to be the low DNA amplification rate and the high detection of contaminating DNA of maternal origin (56). These problems may be successfully resolved in the future by tweaking embryo culture conditions and sample collection. For instance, smaller volumes of media may be used to allow for greater DNA concentration in the culture drop, whilst serial embryo washes prior to specimen collection may ensure the complete disposal of maternal DNA from persisting granulosa cells or degenerating polar bodies. With the aim of an unmanned, uncontaminated and standardized global strategy for molecular analysis of spent culture media, microfluidics technology involving automated media changeover and collection may also be developed and implemented in future incubators. This will allow the establishment of a consistent, reproducible and comprehensive approach to embryo's genome evaluation and embryo selection employable in all IVF treatments without additional workload on the embryology laboratory.

CONCLUSIONS
Recent genomics technological achievements and optimization of embryo culture systems have created a robust PGT methodology, on the base of which solid data and reliable diagnostic conclusions can be generated. This strong analytical platform has indeed enabled the implementation of chromosomes and single gene disorders testing in an increasing portion of IVF treatment cycles, improving clinical outcomes for specific patients populations (i.e., patients at increased risk of generating genetic or chromosomally abnormal embryos). Benefits of chromosomal assessment include the increase in embryo implantation per transfer and an overall reduction in miscarriage rates. These aspects are even more crucial for advanced maternal age patients where majority of embryos are impaired by age-dependent chromosomal abnormalities. Nonetheless, current PGT approaches have margin for improvement, especially for what it concerns parallel chromosomal and single gene testing. Additionally, to ensure that the embryo transferred is free of life-impairing mutations, the mutations tested should not be limited to a specific inheritable one running in the family, but extended into a more comprehensive panel of common conditions, including analysis of de novo mutations occurring in the germline and more common genes/variants related with multifactorial inheritance in the context of ePGT. In the future, supplementary details regarding embryo's reproductive competence and its ability to implant and generate a healthy pregnancy may be acquired by the analysis of additional epigenetic parameters and transcription profiles which might be as well-integrated in the PGT workflow. These complimentary analyses have the potential to further improve clinical outcomes for patients across all ages. However, they may be extensively applied for advanced maternal age patients where success rates are time-sensitive and an accurate evaluation of embryonic development and implantation competence is paramount not only for embryo selection purposes, but also for decisions related to patients future treatment options and chances of success. Although unlikely to be compatible with epigenetic assessment, noninvasive approaches are being investigated for both single gene and chromosomal testing, as well as for discovering alternative markers for embryo paracrine signaling and reproductive competence. Non-invasive approaches represent an extremely interesting field of development for PGT as it would pave the way for its widespread application and full integration into the IVF treatment.
Nonetheless, similarly to other novel genetic testing strategies, the technical ability to perform molecular analyses has occurred at a faster rate than the evidence required for optimal integration could be comprehensively collected and validated. For this reason, it will be crucial for all novel applications discussed in this review to be fully tested, optimized and monitored before they can be offered to patients in a clinical context.

AUTHOR CONTRIBUTIONS
MP, LG, MF, MM, VR, CP, DZ, and AC drafted the manuscript. MP and AC revised the manuscript.


text:
The Immunomodulatory Potential of Wharton's Jelly Mesenchymal Stem/Stromal Cells

The benefits attributed to mesenchymal stem/stromal cells (MSC) in cell therapy applications are mainly attributed to the secretion of factors, which exhibit immunomodulatory and anti-inflammatory effects and stimulate angiogenesis. Despite the desirable features such as high proliferation levels, multipotency, and immune response regulation, there are important variables that must be considered. Although presenting similar morphological aspects, MSC collected from different tissues can form heterogeneous cellular populations and, therefore, manifest functional differences. Thus, the source of MSC should be a factor to be considered in the development of novel therapies. The following text presents an updated review of recent research outcomes related to Wharton's jelly mesenchymal stem/stromal cells (WJ-MSC), harvested from umbilical cords and considered novel and potential candidates for the development of cell-based approaches. This text highlights information on how WJ-MSC affect immune responses in comparison with other sources of MSC.

Introduction
Mesenchymal stem/stromal cells (MSC) are increasingly viewed as sources of cell therapy applications due to their known immunomodulatory and anti-inflammatory effects and capacity to stimulate angiogenesis. Despite the desirable features such as high proliferation levels, multipotency, and immune response regulation, there are important variables that must be considered. Although presenting similar morphological aspects, MSC collected from different tissues can form heterogeneous cellular populations and also manifest tissue-specific functional differences. Thus, the source of MSC should be a factor to be considered in the development of novel therapies. The following text presents an updated review of recent research outcomes related to Wharton's jelly mesenchymal stem/stromal cells (WJ-MSC), harvested from umbilical cords and considered novel and potential candidates for the development of cell-based therapies. This text highlights information on how WJ-MSC affect immune responses in comparison with other sources of MSC. Some of the challenges to be addressed in order to overcome hurdles associated with the therapeutic application of these cells are also included.

The Umbilical Cord Is the Source of Wharton's Jelly
Wharton's jelly (WJ) can be generally described as the mucoid connective tissue that encloses the three umbilical vessels, one vein and 2 arteries, being surrounded by a single layer of amniotic epithelial cells, which constitute the human umbilical cord [1]. Recently, the ongoing interest in umbilical cords as a useful source of MSC encouraged further investigation on these tissue structures. WJ is currently divided into three main zones based on their histological appearance: (a) the subamnion with a sparse population of fibroblast-like cells; (b) the intervascular region, a matrix of connective tissue predominantly made from collagen I, which concentrates the greatest proportion of WJ-MSC; and (c) the perivascular layer that surrounds the umbilical vessels ( Figure 1) [2,3]. WJ-MSC derived from different parts of the same umbilical cord are equally valuable sources for use in cell therapy [4]. Of note, WJ-MSC are different from the hematopoietic stem cells found in the umbilical cord blood [5]. Moreover, as other authors already described [3], WJ is seeded by distinct sources of mesenchymal/stromal cells during the embryological development. These cell subsets express not only relevant markers that characterize both WJ-MSC and perivascular cells but possibly also the main source of progenitor cells that populate the WJ [6].

Benefits of Using WJ-MSC
MSC are considered a potential tool for cell therapy. The "gold standard" bone marrow-derived MSC (BM-MSC) are the most used in clinical trials but have shown mixed results [7][8][9][10][11][12]. Furthermore, their use is not always recommended due to the techniques needed to obtain the cell. BM-MSC are isolated from bone marrow aspirate; this is an invasive procedure and painful for the patient and is accompanied by a risk of infection, possibility of donor morbidity, differences in donor age, and still change or loss of in vitro proliferative and differentiation cellular capacity [13,14]. Alternative sources where isolation is easier, like adipose tissue (AT) and WJ [15], should be and are being considered. AT is an autologous source of cells though some concerns like donor age and risk of infection are the same when compared to bone marrow (BM) [16]. Other alternative sources are, for example, dental pulp [17] and menstrual blood (reviewed in [18]), a well-recognized source of MSC known since 2004. The umbilical cord is usually discarded, mitigating the risks associated with the invasive procedures needed to isolate MSC from BM [16]. With few ethical concerns, WJ is considered an easily accessible source of MSC.
WJ-MSC have been compared not only with BM-MSC but also with AT-MSC (adipose tissue-MSC) and MSC derived from menstrual blood [19] and in most cases, show higher proliferative capacity. In addition, WJ-MSC are very young cells derived from a protected neonatal tissue that has suffered less environmental interference, namely, the effects on the tissues resulting from disease history and life style, a fact that helps the acquisition of a more uniform cell cohort, which may favor their therapeutic application. However, the outcome of functional tests in vitro indicates that they too exhibit limited lifespans and variable immune suppression potentials [20][21][22][23]. WJ-MSC are less prone to develop defective functions that can accumulate throughout a cells' lifespan due to aging and the lifetime exposure to environmental factors [24]. It is important to take into account that quality control for these cells should follow specific criteria such as selecting samples from healthy donors of full-term pregnancies, women over 18 years of age, water broken for no longer than 18h, and the expectant mother must have had at least two consultations during pregnancy and should not present fever or infection at time of birth. Maternal serum screening before delivery should include hemoglobin electrophoresis and serology for prevalent viruses and parasites.
Several reports describe MSC as immune privileged or hypoimmunogenic cells, a status likely enhanced by immunologically protected neighbouring sites, the placenta and the foetus itself [25]. In fact, they express low levels of MHC class I and costimulatory CD40, CD80, and CD86. They also lack expression of MHC class II molecules [24,26,27], in spite of the observation of an upregulated HLA-DR expression on BM-MSC after treatment with IFN-γ, but not with TNF-α. Nevertheless, differing from BM-MSC HLA-DR expression, the same authors did not detect the effect on WJ-MSC [28]. WJ-MSC exhibit enhanced expression of immune suppression proteins, notably leukocyte antigen G6 (HLA-G6) known to have an important role in avoiding immune-based responses against the embryo, indoleamine-2,3-dioxygenase (IDO), and prostaglandin E2 (PGE2) [29]. An important point for consideration is the fact that therapeutic applications involving MSC require an initial in vitro expansion step prior to their use and generally hundreds of millions of cells are used per treatment. It has been shown that several passages in vitro leads to a decrease in BM-MSC self-renewal capacity measured by telomere length shortening and increase in senescence markers [30]. Studies usually evaluate the immunomodulatory capacity of MSC from different sources only in early passages, and few data in the current literature is available on their behavior after passaging in vitro until enough numbers of cells are obtained for use in cell therapy [31,32]. One study comparing AD-MSC and BM-MSC from passage 4 to passage 10 showed that they had similar cell morphology, surface marker expression, and immunomodulatory properties, even though gene expression was different [33]. Despite a higher lifetime in vitro, renewal of WJ-MSC ultimately will also lead to cell arrest and replicative senescence and the result will be the loss of stem cell functionality, even though the senescent cells remain alive [34][35][36]. We previously observed [20] that WJ-MSC from different donors exhibited different lifespans, as measured by senescent phenotype, number of passages, and expansion potential. Moreover, each WJ-MSC sample presented a unique behavior, differing in patterns of cytokine mRNA expression and immunomodulatory properties [37]. Thus, we believe that careful evaluation of senescence markers after repeated passaging plus monitoring of the immunosuppressant potential of each harvested cell must be included in quality control before therapeutic use.

Therapeutic Uses Based on the Immunomodulatory Effects of MSC: Comparing WJ-MSC with BM-MSC
When a tissue is damaged, inflammation occurs and tissueresident MSC and even BM-MSC are mobilized to the lesion site [29,38]. Because of their multipotency, it was believed that recruited MSC differentiated into functional cells to replace the damaged ones. However, this occurrence has eluded researchers. Studies using autologous cells mainly from bone marrow and adipose tissue and/or allogeneic cells from umbilical cord blood have shown that after infusion transdifferentiation of MSC into functional cells in tissues rarely occurs if at all [39]. In turn, it has become increasingly clear that in response to an inflammatory milieu, MSC prepare the microenvironment for tissue repair by producing immunoregulatory molecules that modulate the progression of inflammation, releasing growth factors to produce extracellular matrix [40], stimulating the in situ progenitor cells to differentiate and replace lost cells [41], and promoting angiogenesis [42]. The apparent incongruity between the benefit achieved and the lack of differentiation of the recruited MSC into specialized tissue cells has led to the unraveling of the surprising immunosuppressive capacity of MSC from many different sources [43][44][45][46][47][48]. By now, it is well known that the most promising benefits of therapy with MSC occur in patients presenting inflammatory or autoimmune diseases [49,50]. Thus, the MSC immu-nomodulatory effects may play an important role in the improvement of autoimmune diseases like systemic lupus erythematosus [51,52], type 1 diabetes mellitus [53], and multiple sclerosis [54,55]. Ringden and Le Blanc showed that treatment using an allogenic source of MSC from umbilical cord blood (UCB-MSC), not WJ-MSC, was able to reverse partially or totally GVHD in 50% of patients [56]. In addition, the group headed by Krampera et al. [57] and other researchers [45,58,59] sought to unveil the immunomodulatory mechanisms of BM-MSC, confirming their effect on proliferation and antigen-specific responses by T lymphocytes.
WJ-MSC also appear to show a robust immunomodulatory potential [22]. A comparative study using MSC derived from whole human umbilical cord (MC-MSC) WJ-MSC and BM-MSC showed that MC-MSC proliferated faster and survive longer in culture than WJ-MSC; however, they have similar immunomodulatory potential [60]. Another study comparing BM-MSC and WJ-MSC demonstrated that inflammation affects the immune properties of MSC sources in different ways. Priming BM-MSC enhanced the suppression of phytohemagglutinin (PHA) mitogen-stimulated T cells only, whereas IFN-γ-primed WJ-MSC were better suppressors of MLR (mixed lymphocyte reaction) [28]. BM-MSC, WJ-MSC, and AT-MSC were all capable of suppressing T cell proliferation [61,62]. However, high levels of IL-17A were detected in WJ-MSC cocultures, which is one of the key mediators in the treatment of graft-versushost disease [61]. In a murine experimental autoimmune encephalomyelitis (EAE) model, WJ-MSC treated with IFN-γ increased regulatory T (Treg) cell proliferation and decreased the secretion of inflammatory cytokines in EAE mice, reducing the symptoms of the disease [63].
Of note, human fetal bone marrow (FBM-MSC) and WJ-MSC have biological advantages as compared to adult cells [62]. WJ-MSC have a gene expression pattern similar to AT-MSC but not FBM-MSC. Beyond that, genes associated with cell adhesion, proliferation, and immunomodulatory function are increased in WJ-MSC as revealed by gene ontology. WJ-MSC intrinsically overexpress genes involved in neurotrophic support when compared to BM-MSC, which makes WJ-MSC an interesting candidate for cell therapy in neurodegenerative disorders [64].

MSC Exert Comprehensive Effects on Cell-Mediated Immune Responses
MSC can interact with and regulate the activation and function of immune cells, such as T and B lymphocytes [65,66], dendritic cells (DC) [67], and monocytes/macrophages [68]. The effects of MSC on the immune system are generally anti-inflammatory and are achieved by different, but complementary mechanisms. Nicola et al. showed that human BM-MSC are capable of suppressing T cell proliferation in a mixed lymphocyte reaction (MLR) or when T cells are activated by phytohemagglutinin (PHA) [45]. WJ-MSC suppress mitogen-induced T cell responses to a greater extent than either BM-MSC or AT-MSC [28]. Recently, our group showed that different samples of human WJ-MSC were capable of inhibiting mitogenactivated CD3+ T cell proliferation, although to different extents, though the immunomodulatory profile of each WJ-MSC was essentially maintained even after 10 passages [37]. Another mechanism involved in immune suppression is T cell anergy. BM-MSC can induce T cell anergy by suppressing cyclin D2 expression and inhibiting CD4+ and CD8+ T cell proliferation by producing nitric oxide [69,70]. BM-MSC are also capable of regulating the immune response by the induction of Treg, and it has been reported that they can induce T cell apoptosis via the Fas/FasL pathway. The apoptotic cells will stimulate macrophages to secrete high levels of TGF-β, which in turn will generate Treg cells [71]. Our preliminary results (unpublished data) showed that WJ-MSC were also able to induce Treg cells when cocultured with PBMC and treated with IFN-γ. BM-MSC also affect B cell functions, inhibiting the proliferation of activated B cells, their antibody production, and their chemotactic behavior [72]. BM-MSC have been shown to interfere in differentiation, maturation, and function of DC [67]. For example, in coculture, DC lose their ability to induce T cell activation [73][74][75]. Likewise, the differentiation of monocytes into mature DCs was inhibited and costimulatory ligand expression was blocked when cultured with WJ-MSC [76]. Taken together, the available literature indicates that WJ-MSC possess immunological features comparable to the better studied BM-MSC and even to MSC from other sources, but further detailing is needed to find the best therapeutic indications for this allogeneic source of cells as a substitute for the autologous BM-MSC and AT-MSC. The fact that WJ-MSC constitute an allogeneic therapy may in fact favor these cells in certain pathologies where the immunosuppressive response is urgent and should encompass cell and humoral responses.
An additional twist in this rationale is the observation that MSC, both in vitro and in vivo, seem capable of adopting a pro-or anti-inflammatory phenotype. Similar to the phenotype-switching phenomenon in macrophages massively explored throughout the literature and reviewed elsewhere [77,78], MSC are also sensitive to shifts in the local immune milieu. Disruption towards an excessive concentration of proinflammatory cytokines such as IFN-γ and TNF-α activates signalling pathways by way of sensors present on human BM-MSC, causing a shift to the MSC2 phenotype and playing an important role in the downregulation of immune cells and their corresponding proinflammatory mediators [79]. In contrast, to switch to a MSC1-type profile, an anti-inflammatory microenvironment is required and MSC1 will not only express lower levels of immunosuppressive genes including IDO, NO, and PGE2 but will also be a major source of proinflammatory molecules, which will recruit and activate immune cells by secreting IL-6 and producing IL-1α and IL-1β [78]. BM-MSC, under conditions of hypoxia and stimulated with proinflammatory cytokines such as IFN-γ, TNF-α, and IL-1β, increase the expression of Toll-like receptors TLR2, TLR3, and TLR4, rendering these cells more sensitive to the inflammatory medium [80]. Waterman et al. showed that BM-MSC acquired two distinct phenotypes after stimulation with TLR3 and TLR4 ligands and accordingly, resulted in different immunomodu-latory effects. Indeed, LPS-stimulated BM-MSC (TLR4 ligand) exhibited a proinflammatory profile (MSC1) in contrast with the polyI:C stimulated BM-MSC (TLR3 ligand) that showed an anti-inflammatory profile (MSC2) [79]. The same group also showed that BM-MSC induced into expressing the MSC1 profile attenuate cancer cell growth while when the same cells exhibit a MSC2 phenotype, they act similarly to conventional MSC in promoting tumor growth and metastases [81].
The bottom-line result of MSC switching to a type 1 profile is ultimately an overall immune modulation opposing the local environment [78]. In an inflammatory milieu, the induction of a type 2 MSC will lead to the regulation of excessive immune responses at the focal point of injury, the desirable scenario to heal damaged tissue, sponsored and facilitated by MSC plasticity.

Conflicts of Interest
The authors declare that they have no conflicts of interest.