The Genetic Discrimination Observatory: confronting novel issues in genetic discrimination.

Genetic discrimination (GD) is the differential or unfair profiling of an individual on the basis of genetic data. This article summarizes the actions of the Genetic Discrimination Observatory (GDO) in addressing GD and recent developments in GD since late 2020. It shows how GD can take many forms in today's rapidly evolving society.

Comparative Molecular Genetics of Odontogenic Keratocysts in Sporadic and Syndromic Patients.

Odontogenic keratocysts (OKCs) are common cysts of odontogenic origin that usually occur as a single nonsyndromic cyst in isolation (sporadic) or as syndromic multiple cysts as a manifestation of naevoid basal cell carcinoma syndrome. Alterations involving the PTCH gene are the most commonly identified factor associated with up to 85% and 84% of naevoid basal cell carcinoma syndrome and sporadic cases, respectively. Other Hedgehog pathway and non-Hedgehog pathway-associated genes have been implicated in the pathogenesis of OKCs. This pilot study used the Affymetrix OncoScan molecular assay to perform a comparative genomic analysis between 4 sporadic and 3 syndromic cases of OKC to identify molecular drivers that may be common and/or distinct in these 2 groups. The majority of alterations detected in both groups were copy number neutral loss of heterozygosity. Despite distinct molecular signatures observed in both groups, copy number neutral loss of heterozygosity alterations involving chromosome 9q affecting not only PTCH but also the NOTCH1 gene were detected in all syndromic and 3 sporadic cases. Loss of heterozygosity alterations involving 16p11.2 affecting genes not previously described in OKCs were also detected in all syndromic and 3 sporadic cases. Furthermore, alterations on 22q11.23 and 10q22.1 were also detected in both groups. Of note, alterations on 1p13.3, 2q22.1, and 6p21.33 detected in sporadic cases were absent in all syndromic cases. This study demonstrates that a more common group of genes may be affected in both groups of OKCs, whereas other alterations may be useful in distinguishing sporadic from syndromic cysts. These findings should be validated in larger OKC cohorts to improve molecular diagnosis and subsequent patient management.

Effect of 2-methoxyestradiol treatment on early- and late-stage breast cancer progression in a mouse model.

The prevalence of breast cancer (BC) continues to increase and is the leading cause of cancer deaths in many countries. Numerous in vitro and in vivo studies have demonstrated that 2-methoxyestradiol (2-ME) has antiproliferative and antiangiogenic effects in BC, thereby inhibiting tumour growth and metastasis. We compared the effect of 2-ME in early- and late-stage BC using a transgenic mouse model-FVB/N-Tg(MMTV-PyVT)-of spontaneously development of aggressive mammary carcinoma with lung metastasis. Mice received 100 mg/kg 2-ME treatment immediately when palpable mammary tumours were identified (early-stage BC; Experimental group 1) and 28 days after palpable mammary tumours were detected (late-stage BC; Experimental group 2). 2-ME was administered via oral gavage three times a week for 28 days after initiation of treatment, whereas control mice received the vehicle containing 10% dimethyl sulfoxide and 90% sunflower oil for the same duration as the treatment group. Mammary tumours were measured weekly over the 28 days and at termination, blood, mammary and lung tissue were collected for analysis. Mice with a tumour volume threshold of 4000 mm3 were killed before the treatment regime was completed. 2-ME treatment of early-stage BC led to lower levels of mammary tumour necrosis, whereas tumour mass and volume were increased. Additionally, necrotic lesions and anti-inflammatory CD163-expressing cells were more frequent in pulmonary metastatic tumours in this group. In contrast, 2-ME treatment of late-stage BC inhibited tumour growth over the 28-day period and resulted in increased CD3+ cell number and tumour necrosis. Furthermore, 2-ME treatment slowed down pulmonary metastasis but did not increase survival of late-stage BC mice. Besides late-stage tumour necrosis, none of the other results were statistically significant. This study demonstrates that 2-ME treatment has an antitumour effect on late-stage BC, however, with no increase in survival rate, whereas the treatment failed to demonstrate any benefit in early-stage BC.

Characteristics and outcomes of neonates with intrapartum asphyxia managed with therapeutic hypothermia in a public tertiary hospital in South Africa.

BACKGROUND: In randomized clinical trials, therapeutic hypothermia (TH) has been shown to reduce death and/or moderate-to-severe disability in neonates with hypoxic ischemic encephalopathy (HIE) in high-income countries, while this has not consistently been the case in low-and middle-income countries (LMICs). Many studies reporting on outcomes of neonates with HIE managed with TH are those conducted under controlled study conditions, and few reporting in settings where this intervention is offered as part of standard of care, especially from LMICs. In this study we report on short-term outcomes of neonates with moderate-to-severe HIE where TH was offered as part of standard of care. OBJECTIVE: To determine characteristics and mortality rate at hospital discharge in neonates with moderate-to-severe HIE. METHODS: Hospital records of neonates with intrapartum asphyxia were reviewed for clinical findings, management with TH (cooled or non-cooled) and mortality at hospital discharge. Inclusion criteria were birthweight ≥ 1800 g, gestational age ≥ 36 weeks and moderate-to-severe HIE. Comparisons were made between survivors and non-survivors in cooled and/or non-cooled neonates. RESULTS: Intrapartum asphyxia was diagnosed in 856 neonates, with three having no recorded HIE status; 30% (258/853) had mild HIE, and 595/853 (69%) with moderate-to-severe HIE. The overall incidence of intrapartum asphyxia was 8.8/1000 live births. Of the 595 with moderate-to-severe HIE, three had no records on cooling and 67% (399/592) were cooled. Amongst 193 non-cooled neonates, 126 (67%) had documented reasons for not being cooled with common reasons being a moribund neonate (54.0%), equipment unavailability (11.1%), pulmonary hypertension (9.5%), postnatal age > 6 h on admission (8.7%), and improvement in severity of encephalopathy (8.7%). Overall mortality was 29.0%, being 17.0% and 53.4% in cooled and non-cooled infants respectively. On multivariate analysis, the only factor associated with mortality was severe encephalopathy. CONCLUSION: Overall mortality in neonates with moderate-to-severe HIE was 29.0% and 17.0% in those who were cooled. Cooling was not offered to all neonates mainly because of severe clinical illness, equipment unavailability and delayed presentation, making it difficult to assess overall impact of this intervention. Prospective clinical studies need to be conducted in LMIC to further assess effect of TH in short and long-term outcomes.

Genetic Associations with Coronavirus Susceptibility and Disease Severity.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) global public health emergency, and the disease it causes is highly variable in its clinical presentation. Host genetic factors are increasingly recognised as a determinant of infection susceptibility and disease severity. Several initiatives and groups have been established to analyse and review host genetic epidemiology associated with COVID-19 outcomes. Here, we review the genetic loci associated with COVID-19 susceptibility and severity focusing on the common variants identified in genome-wide association studies.

The NESHIE and CP Genetics Resource (NCGR): A database of genes and variants reported in neonatal encephalopathy with suspected hypoxic ischemic encephalopathy (NESHIE) and consequential cerebral palsy (CP).

Neonatal encephalopathy (NE) with suspected hypoxic ischaemic encephalopathy (HIE) (NESHIE) is a complex syndrome occurring in newborns, characterised by altered neurological function. It has been suggested that genetic variants may influence NESHIE susceptibility and outcomes. Unlike NESHIE, for which a limited number of genetic studies have been performed, many studies have identified genetic variants associated with cerebral palsy (CP), which can develop from severe NESHIE. Identifying variants in patients with CP, as a consequence of NESHIE, may provide a starting point for the identification of genetic variants associated with NESHIE outcomes. We have constructed NCGR (NESHIE and CP Genetics Resource), a database of genes and variants reported in patients with NESHIE and CP (where relevant to NESHIE), for the purpose of collating and comparing genetic findings between the two conditions. In this paper we describe the construction and functionality of NCGR. Furthermore, we demonstrate how NCGR can be used to prioritise genes and variants of potential clinical relevance that may underlie a genetic predisposition to NESHIE and contribute to an understanding of its pathogenesis.

Perspectives on establishing a public cord blood inventory in South Africa.

The South African population is highly diverse, both ethnically and genetically. This diversity is particularly true for the African ancestry and various mixed ancestry population groups. These groups are under-represented in national and international bone marrow and peripheral blood donor registries, making it challenging to identify HLA-matched and mismatched unrelated donors when patients from these groups require allogeneic hematopoietic stem and progenitor cell transplantation. In most high-income countries, banked cord blood (CB) units provide an attractive source of hematopoietic progenitor cells for genetically diverse populations. SA does not have a public CB inventory, leaving many patients without access to this important treatment modality. Haploidentical transplantation provides an alternative. In recent years, the use of post-transplant cyclophosphamide has significantly reduced the incidence of graft-versus-host disease after haploidentical transplantation and has improved transplantation outcomes. However, it is difficult to identify suitable haploidentical donors in SA because of family disruption and a high prevalence of HIV. Here the authors provide a brief historical overview of the ethnic and genetic diversity of the country and region. The authors provide a southern African perspective on HLA diversity, consider the allogeneic hematopoietic stem and progenitor cell transplantation landscape and explore the need to establish a public CB bank (CBB) in SA. The health policy and regulatory frameworks that will impact on a CBB in the country SA are also explored. Finally, the authors discuss several matters we believe require attention when considering the establishment of a sustainable public CBB in the South African context.

Characterization of Signalling Pathways That Link Apoptosis and Autophagy to Cell Death Induced by Estrone Analogues Which Reversibly Depolymerize Microtubules.

The search for novel anti-cancer compounds which can circumvent chemotherapeutic drug resistance and limit systemic toxicity remains a priority. 2-Ethyl-3-O-sulphamoyl-estra-1,3,5(10)15-tetraene-3-ol-17one (ESE-15-one) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16) are sulphamoylated 2-methoxyestradiol (2-ME) analogues designed by our research team. Although their cytotoxicity has been demonstrated in vitro, the temporal and mechanistic responses of the initiated intracellular events are yet to be determined. In order to do so, assays investigating the compounds' effects on microtubules, cell cycle progression, signalling cascades, autophagy and apoptosis were conducted using HeLa cervical- and MDA-MB-231 metastatic breast cancer cells. Both compounds reversibly disrupted microtubule dynamics as an early event by binding to the microtubule colchicine site, which blocked progression through the cell cycle at the G1/S- and G2/M transitions. This was supported by increased pRB and p27Kip1 phosphorylation. Induction of apoptosis with time-dependent signalling involving the p-JNK, Erk1/2 and Akt/mTOR pathways and loss of mitochondrial membrane potential was demonstrated. Inhibition of autophagy attenuated the apoptotic response. In conclusion, the 2-ME analogues induced a time-dependent cross-talk between cell cycle checkpoints, apoptotic signalling and autophagic processes, with an increased reactive oxygen species formation and perturbated microtubule functioning appearing to connect the processes. Subtle differences in the responses were observed between the two compounds and the different cell lines.

Lessons Learned from Somatic Cell Nuclear Transfer.

Somatic cell nuclear transfer (SCNT) has been an area of interest in the field of stem cell research and regenerative medicine for the past 20 years. The main biological goal of SCNT is to reverse the differentiated state of a somatic cell, for the purpose of creating blastocysts from which embryonic stem cells (ESCs) can be derived for therapeutic cloning, or for the purpose of reproductive cloning. However, the consensus is that the low efficiency in creating normal viable offspring in animals by SCNT (1-5%) and the high number of abnormalities seen in these cloned animals is due to epigenetic reprogramming failure. In this review we provide an overview of the current literature on SCNT, focusing on protocol development, which includes early SCNT protocol deficiencies and optimizations along with donor cell type and cell cycle synchrony; epigenetic reprogramming in SCNT; current protocol optimizations such as nuclear reprogramming strategies that can be applied to improve epigenetic reprogramming by SCNT; applications of SCNT; the ethical and legal implications of SCNT in humans; and specific lessons learned for establishing an optimized SCNT protocol using a mouse model.

Adipogenesis: A Complex Interplay of Multiple Molecular Determinants and Pathways.

The formation of adipocytes during embryogenesis has been largely understudied. However, preadipocytes appear to originate from multipotent mesenchymal stromal/stem cells which migrate from the mesoderm to their anatomical localization. Most studies on adipocyte formation (adipogenesis) have used preadipocytes derived from adult stem/stromal cells. Adipogenesis consists of two phases, namely commitment and terminal differentiation. This review discusses the role of signalling pathways, epigenetic modifiers, and transcription factors in preadipocyte commitment and differentiation into mature adipocytes, as well as limitations in our understanding of these processes. To date, a limited number of transcription factors, genes and signalling pathways have been described to regulate preadipocyte commitment. One reason could be that most studies on adipogenesis have used preadipocytes already committed to the adipogenic lineage, which are therefore not suitable for studying preadipocyte commitment. Conversely, over a dozen molecular players including transcription factors, genes, signalling pathways, epigenetic regulators, and microRNAs have been described to be involved in the differentiation of preadipocytes to adipocytes; however, only peroxisome proliferator-activated receptor gamma has proven to be clinically relevant. A detailed understanding of how the molecular players underpinning adipogenesis relate to adipose tissue function could provide new therapeutic approaches for addressing obesity without compromising adipose tissue function.

The Role of Pref-1 during Adipogenic Differentiation: An Overview of Suggested Mechanisms.

Obesity contributes significantly to the global health burden. A better understanding of adipogenesis, the process of fat formation, may lead to the discovery of novel treatment strategies. However, it is of concern that the regulation of adipocyte differentiation has predominantly been studied using the murine 3T3-L1 preadipocyte cell line and murine experimental animal models. Translation of these findings to the human setting requires confirmation using experimental models of human origin. The ability of mesenchymal stromal/stem cells (MSCs) to differentiate into adipocytes is an attractive model to study adipogenesis in vitro. Differences in the ability of MSCs isolated from different sources to undergo adipogenic differentiation, may be useful in investigating elements responsible for regulating adipogenic differentiation potential. Genes involved may be divided into three broad categories: early, intermediate and late-stage regulators. Preadipocyte factor-1 (Pref-1) is an early negative regulator of adipogenic differentiation. In this review, we briefly discuss the adipogenic differentiation potential of MSCs derived from two different sources, namely adipose-derived stromal/stem cells (ASCs) and Wharton's Jelly derived stromal/stem cells (WJSCs). We then discuss the function and suggested mechanisms of action of Pref-1 in regulating adipogenesis, as well as current findings regarding Pref-1's role in human adipogenesis.

Comparison of human platelet lysate alternatives using expired and freshly isolated platelet concentrates for adipose-derived stromal cell expansion.

Pooled human platelet lysate (pHPL) has been used to expand adipose-derived stromal cells (ASCs) and can be formulated using fresh or expired buffy coats (BCs) which are then resuspended in either plasma or an additive solution. Not much is known about the effects that expired products and additive solutions have on ASC expansion, and the need for quality control and release criteria has been expressed. This pilot study compared proliferation, cell size, morphology and immunophenotype of ASCs expanded in the different pHPL alternatives versus foetal bovine serum (FBS). Quality control criteria were assessed prior to and during the manufacture of the pHPL alternatives. ASCs were then expanded in 1%, 2.5%, 5% or 10% of the different pHPL alternatives or in 10% FBS. Cell size, morphology, cell number and immunophenotype were measured using microscopy and flow cytometry. The majority of the pHPL alternatives were within the recommended ranges for the quality control criteria. ASCs expanded in the pHPL alternatives were smaller in size, displayed a tighter spindle-shaped morphology, increased cell growth and had a similar immunophenotype (with the exception of CD34 and CD36) when compared to ASCs expanded in FBS. Here we report on the effects that expired BC products and additive solutions have on ASC expansion. When taken together, our findings indicate that all of the pHPL alternatives can be considered to be suitable replacements for FBS for ASC expansion, and that expired BC products can be used as an alternative to fresh BC products.

The Role of Reactive Oxygen Species in Adipogenic Differentiation.

Interest in reactive oxygen species and adipocyte differentiation/adipose tissue function is steadily increasing. This is due in part to a search for alternative avenues for combating obesity, which results from the excess accumulation of adipose tissue. Obesity is a major risk factor for complex disorders such as cancer, type 2 diabetes, and cardiovascular diseases. The ability of mesenchymal stromal/stem cells (MSCs) to differentiate into adipocytes is often used as a model for studying adipogenesis in vitro. A key focus is the effect of both intra- and extracellular reactive oxygen species (ROS) on adipogenesis. The consensus from the majority of studies is that ROS, irrespective of the source, promote adipogenesis.The effect of ROS on adipogenesis is suppressed by antioxidants or ROS scavengers. Reactive oxygen species are generated during the process of adipocyte differentiation as well as by other cell metabolic processes. Despite many studies in this field, it is still not possible to state with certainty whether ROS measured during adipocyte differentiation are a cause or consequence of this process. In addition, it is still unclear what the exact sources are of the ROS that initiate and/or drive adipogenic differentiation in MSCs in vivo. This review provides an overview of our understanding of the role of ROS in adipocyte differentiation as well as how certain ROS scavengers and antioxidants might affect this process.

Reactive Oxygen Species and NOX Enzymes Are Emerging as Key Players in Cutaneous Wound Repair.

Our understanding of the role of oxygen in cell physiology has evolved from its long-recognized importance as an essential factor in oxidative metabolism to its recognition as an important player in cell signaling. With regard to the latter, oxygen is needed for the generation of reactive oxygen species (ROS), which regulate a number of different cellular functions including differentiation, proliferation, apoptosis, migration, and contraction. Data specifically concerning the role of ROS-dependent signaling in cutaneous wound repair are very limited, especially regarding wound contraction. In this review we provide an overview of the current literature on the role of molecular and reactive oxygen in the physiology of wound repair as well as in the pathophysiology and therapy of chronic wounds, especially under ischemic and hyperglycemic conditions.

The Role of Tumor Microenvironment in Chemoresistance: To Survive, Keep Your Enemies Closer.

Chemoresistance is a leading cause of morbidity and mortality in cancer and it continues to be a challenge in cancer treatment. Chemoresistance is influenced by genetic and epigenetic alterations which affect drug uptake, metabolism and export of drugs at the cellular levels. While most research has focused on tumor cell autonomous mechanisms of chemoresistance, the tumor microenvironment has emerged as a key player in the development of chemoresistance and in malignant progression, thereby influencing the development of novel therapies in clinical oncology. It is not surprising that the study of the tumor microenvironment is now considered to be as important as the study of tumor cells. Recent advances in technological and analytical methods, especially 'omics' technologies, has made it possible to identify specific targets in tumor cells and within the tumor microenvironment to eradicate cancer. Tumors need constant support from previously 'unsupportive' microenvironments. Novel therapeutic strategies that inhibit such microenvironmental support to tumor cells would reduce chemoresistance and tumor relapse. Such strategies can target stromal cells, proteins released by stromal cells and non-cellular components such as the extracellular matrix (ECM) within the tumor microenvironment. Novel in vitro tumor biology models that recapitulate the in vivo tumor microenvironment such as multicellular tumor spheroids, biomimetic scaffolds and tumor organoids are being developed and are increasing our understanding of cancer cell-microenvironment interactions. This review offers an analysis of recent developments on the role of the tumor microenvironment in the development of chemoresistance and the strategies to overcome microenvironment-mediated chemoresistance. We propose a systematic analysis of the relationship between tumor cells and their respective tumor microenvironments and our data show that, to survive, cancer cells interact closely with tumor microenvironment components such as mesenchymal stem cells and the extracellular matrix.

Novel flow cytometric approach for the detection of adipocyte subpopulations during adipogenesis.

The ability of mesenchymal stromal cells (MSCs) to differentiate into adipocytes provides a cellular model of human origin to study adipogenesis in vitro. One of the major challenges in studying adipogenesis is the lack of tools to identify and monitor the differentiation of various subpopulations within the heterogeneous pool of MSCs. Cluster of differentiation (CD)36 plays an important role in the formation of intracellular lipid droplets, a key characteristic of adipocyte differentiation/maturation. The objective of this study was to develop a reproducible quantitative method to study adipocyte differentiation by comparing two lipophilic dyes [Nile Red (NR) and Bodipy 493/503] in combination with CD36 surface marker staining. We identified a subpopulation of adipose-derived stromal cells that express CD36 at intermediate/high levels and show that combining CD36 cell surface staining with neutral lipid-specific staining allows us to monitor differentiation of adipose-derived stromal cells that express CD36(intermediate/high)during adipocyte differentiation in vitro. The gradual increase of CD36(intermediate/high/)NR(positive)cells during the 21 day adipogenesis induction period correlated with upregulation of adipogenesis-associated gene expression.

Lentivector Knockdown of CCR5 in Hematopoietic Stem and Progenitor Cells Confers Functional and Persistent HIV-1 Resistance in Humanized Mice.

UNLABELLED: Gene-engineered CD34(+) hematopoietic stem and progenitor cells (HSPCs) can be used to generate an HIV-1-resistant immune system. However, a certain threshold of transduced HSPCs might be required for transplantation into mice for creating an HIV-resistant immune system. In this study, we combined CCR5 knockdown by a highly efficient microRNA (miRNA) lentivector with pretransplantation selection of transduced HSPCs to obtain a rather pure population of gene engineered CD34(+) cells. Low-level transduction of HSPCs and subsequent sorting by flow cytometry yielded >70% transduced cells. Mice transplanted with these cells showed functional and persistent resistance to a CCR5-tropic HIV strain: viral load was significantly decreased over months, and human CD4(+) T cells were preserved. In one mouse, viral mutations, resulting presumably in a CXCR4-tropic strain, overcame HIV resistance. Our results suggest that HSPC-based CCR5 knockdown may lead to efficient control of HIV in vivo. We overcame a major limitation of previous HIV gene therapy in humanized mice in which only a proportion of the cells in chimeric mice in vivo are anti-HIV engineered. Our strategy underlines the promising future of gene engineering HIV-resistant CD34(+) cells that produce a constant supply of HIV-resistant progeny. IMPORTANCE: Major issues in experimental long-term in vivo HIV gene therapy have been (i) low efficacy of cell transduction at the time of transplantation and (ii) transduction resulting in multiple copies of heterologous DNA in target cells. In this study, we demonstrated the efficacy of a transplantation approach with a selection step for transduced cells that allows transplantation of an enriched population of HSPCs expressing a single (low) copy of a CCR5 miRNA. Efficient maintenance of CD4(+) T cells and a low viral titer resulted only when at least 70% of the HIV target cells were genetically modified. These findings imply that clinical protocols of HIV gene therapy require a selective enrichment of genetically targeted cells because positive selection of modified cells is likely to be insufficient below this threshold. This selection approach may be beneficial not only for HIV patients but also for other patients requiring transplantation of genetically modified cells.

Cystic fibrosis on the African continent.

Cystic fibrosis (CF; OMIM 219700) is a life-shortening and costly autosomal recessive disease that has been most extensively studied in individuals of Caucasian descent. There is ample evidence, however, that it also affects other ethnicities. In Africa there have been several reports of CF, but there has been no concerted effort toward establishing the molecular epidemiology of this disease on the continent, which is the first step toward outlining a public health strategy to effectively address the needs of these patients. A literature search revealed reports from only 12 of the 54 African states on the molecular analysis of the mutations present in suspected CF patients, resulting in the identification of 79 mutations. Based on previous functional investigations, 39 of these cause CF, 10 are of varying clinical consequence, 4 have no associated evidence regarding whether they cause CF, 4 are synonymous, 5 are novel, and 21 are unique to Africa. We propose that CF be more thoroughly investigated on the continent to ensure that the public health needs of African CF patients-both those in Africa and those of African descent living elsewhere-are met.Genet Med 18 7, 653-662.

Fibroblast-Derived Extracellular Matrix Induces Chondrogenic Differentiation in Human Adipose-Derived Mesenchymal Stromal/Stem Cells in Vitro.

Mesenchymal stromal/stem cells (MSCs) represent an area being intensively researched for tissue engineering and regenerative medicine applications. MSCs may provide the opportunity to treat diseases and injuries that currently have limited therapeutic options, as well as enhance present strategies for tissue repair. The cellular environment has a significant role in cellular development and differentiation through cell-matrix interactions. The aim of this study was to investigate the behavior of adipose-derived MSCs (ad-MSCs) in the context of a cell-derived matrix so as to model the in vivo physiological microenvironment. The fibroblast-derived extracellular matrix (fd-ECM) did not affect ad-MSC morphology, but reduced ad-MSC proliferation. Ad-MSCs cultured on fd-ECM displayed decreased expression of integrins α2 and ß1 and subsequently lost their multipotency over time, as shown by the decrease in CD44, Octamer-binding transcription factor 4 (OCT4), SOX2, and NANOG gene expression. The fd-ECM induced chondrogenic differentiation in ad-MSCs compared to control ad-MSCs. Loss of function studies, through the use of siRNA and a mutant Notch1 construct, revealed that ECM-mediated ad-MSCs chondrogenesis requires Notch1 and ß-catenin signaling. The fd-ECM also showed anti-senescence effects on ad-MSCs. The fd-ECM is a promising approach for inducing chondrogenesis in ad-MSCs and chondrogenic differentiated ad-MSCs could be used in stem cell therapy procedures.