Neonatal encephalopathy suspected to be hypoxic-ischaemic encephalopathy (NESHIE) remains a leading cause of neonatal mortality and long-term neurodevelopmental impairment, particularly in low- and middle-income countries. While therapeutic hypothermia reduces mortality in moderate to severe cases, a significant proportion of affected infants continue to experience adverse neurological outcomes. This multi-centre observational study aims to elucidate the clinical and biological mechanisms underlying NESHIE by conducting a comprehensive comparative analysis of neonates with moderate to severe NESHIE and healthy term controls. Participants with NESHIE were previously recruited under an existing approved protocol (University of Pretoria ethics reference: 481/2017), and healthy neonates will be newly enrolled. The study will integrate clinical and molecular data to: (1) identify clinical risk factors associated with NESHIE; (2) perform whole genome sequencing to detect relevant genetic variants; (3) analyse DNA methylation patterns via bisulfite sequencing; (4) assess gene expression using bulk and single-cell RNA sequencing (RNA-seq); (5) characterise proteomic and metabolomic profiles through liquid chromatography-mass spectrometry of dried blood spot samples; (6) examine the placental microbiome; and (7) evaluate placental histopathological differences between groups. By offering a multi-dimensional view of the molecular and microbial landscape of NESHIE in a South African cohort, this study aims to enhance understanding of the disease pathogenesis. Ultimately, the findings may support the development of biomarkers for early diagnosis, improve risk stratification, and guide novel therapeutic strategies for affected neonates. The study has received National Health Research Database (NHRD) registration under GP_202411_053 (Gauteng) and WC_202411_026 (Western Cape), with ethics approvals granted by the University of Pretoria (184/2024), University of the Witwatersrand (250406B), and Stellenbosch University (N24/12/154_RECIP_UP184/2024) as well as their respective tertiary academic hospitals.

Obesity is increasingly recognised as an important factor contributing to cancer progression, particularly in breast cancer. However, the cellular and molecular mechanisms underlying obesity-driven breast cancer remain unclear. Several theories have been proposed to explain the complex interactions between adipose tissue and cancer, incorporating both local and distant crosstalk. This review describes existing theories on the cellular and molecular mechanisms linking obesity and breast cancer progression with a focus on inflammation, oxidative stress, metabolic reprogramming, the tumour microenvironment and crosstalk between adipose tissue and tumours. The nature of these interactions appears to be influenced by the specific characteristics of adipose tissue, including its type and anatomical location, which play distinct roles in modulating breast cancer risk and outcomes. Understanding the cellular and molecular mechanisms that underlie obesity-driven breast cancer progression may pave the way for the development of targeted therapies.

Mesenchymal stromal/stem cells (MSCs) possess unique immunomodulatory and regenerative properties, making them ideal candidates for addressing a variety of disorders. With an increasing number of clinical trials, reliable sources are needed to obtain sufficient cell numbers and to circumvent inherent limitations associated with primary cells. Induced pluripotent stem cells (iPSCs) can be differentiated into various somatic cell types and are able to proliferate indefinitely in culture. Differentiation of iPSCs presents a viable alternative in order to create target cell types for both proof-of-concept research and therapeutic applications. In this chapter, we present a protocol to derive MSC-like cells from iPSCs as well as methods of characterizing these cells.

Regenerative medicine is a relatively new branch of therapeutics in equine medicine, which aims to restore and reconstitute tissue function and structure via cellular and/or noncellular approaches. Biological constituents such as mesenchymal stromal/stem cells (MSCs) are potent therapeutics, which can aid in damaged tissue regeneration due to their differentiation capacity into many different cell types such as adipose tissue, bone, and cartilage. MSCs can be successfully and conveniently isolated from equine subcutaneous adipose tissue (adipose-derived stromal cells, ASCs). In horses, there are currently no standardized methods for characterizing ASCs both in research and clinical practice. However, many studies are investigating antibodies which may be expressed on the ASC cell surface through multicolor immunophenotyping. This chapter describes techniques used to isolate, grow, characterize, and store equine ASCs, which have been adapted from extensive published research from the Institute of Cellular and Molecular Medicine (ICMM) at the University of Pretoria.

Neonatal encephalopathy with suspected hypoxic ischaemic encephalopathy (NESHIE) is a neurological disorder caused by oxygen deprivation and limited blood flow to a neonate’s brain. Although various antenatal and perinatal factors have been identified, their precise role in NESHIE pathogenesis remains unclear. The pathophysiology involves multiple molecular pathways that can be explored using a multi-omics approach, including epigenetics. Epigenetics involves heritable changes in gene expression without altering the DNA sequence, encompassing chemical modifications to DNA and histone proteins, as well as changes mediated by non-coding RNAs (ncRNAs). These epigenetic changes regulate gene expression and can be influenced by environmental factors, offering crucial insights into gene regulation and disease mechanisms. This review examines the role of epigenetic mechanisms in NESHIE, focusing on the modulation of hypoxia-inducible factor-1 alpha (HIF-1α) and ncRNA during hypoxic conditions. Additionally, epigenetic-mediated foetal programming may shed light on how maternal and antenatal risk factors contribute to NESHIE susceptibility. Understanding these epigenetic signatures could advance biomarker discovery and the development of novel therapeutic strategies for NESHIE.

BACKGROUND:The role of mesenchymal stromal/stem cells (MSCs) in tumour development and progression remains a subject of debate. Previous studies have reported contradictory outcomes, possibly due to variations in experimental design and the use of xenograft models. Xenograft models limit interpretation and translation due to cross-species variability. To address these limitations, we employed an isogenic mouse model of spontaneous breast cancer (BC) to investigate the impact of murine MSCs on BC development and progression. METHODS:MSCs isolated from FVB/N mouse adipose tissue (mASCs) were administered to female mice with palpable mammary tumours. Tumour volume and mass were assessed, and analysis of histopathological necrosis and gene expression was conducted on mammary (MT) and lung metastatic tumours (LT). RESULTS:No change in MT mass and volume was observed between mASC-treated and control mice. However, mASC treatment led to increased necrosis in LT but not in MT. Immunohistochemistry revealed that mASC-treated mice had fewer CD163+ anti-inflammatory macrophages in the LT but not in the MT. Tgf-β3, vegfr1, and cd105 were observed and downregulated in both MT and LT in mASC-treated mice. The downregulation of cd36 and tgf-β3 contributes to pro-tumourigenic activities, whereas the downregulation of vegfr1 and cd105 is associated with an anti-tumour effect. In the mASC treatment group, all cytokines tested for, except IL-27, were elevated. CONCLUSION:This study suggests that mASCs are anti-tumourigenic in pulmonary metastatic BC. Our findings emphasize the importance of considering the tumour microenvironment and employing relevant animal models when investigating the impact of MSCs on tumour progression.

Reactive oxygen species (ROS) play an important role in the differentiation of immature stem cells to mature adipocytes in a process known as adipogenesis. Despite decades of study, there is still a gap in our understanding of the exact mechanisms played by ROS in regulating adipogenesis. This chapter describes flow cytometric measurement of endogenous ROS production during adipogenesis in adipose-derived stromal/stem cells (ASCs).

Adipogenesis is a complex, multistep process by which precursor cells such as mesenchymal stromal/stem cells (MSCs) differentiate into mature adipocytes. The ability of MSCs to differentiate into adipocytes serves as a model to study human adipogenesis in vitro. Flow cytometry is a powerful technique that can be used to analyze the properties of individual cells. In this chapter, we describe a flow cytometry-based technique to quantify the proportion of MSCs that have differentiated into adipocytes.

Adipose-derived mesenchymal stromal/stem cells (AD-MSCs) offer a promising cell source for regenerative medicine due to their easy accessibility and potential application across a wide range of disorders. This chapter describes a standardized enzymatic isolation protocol to extract AD-MSCs from both solid adipose tissue and lipoaspirate. Adipose tissue samples are initially washed with phosphate-buffered saline (PBS) to remove blood contaminants. Tissue is then digested enzymatically with type I collagenase under controlled temperature and agitation to release the stromal vascular fraction (SVF) containing AD-MSCs. The enzyme activity is neutralized using complete growth medium followed by centrifugation to separate the cell pellet from unwanted material. The resulting cell pellet is resuspended and filtered to eliminate residual tissue fragments. After seeding the cells in culture, AD-MSCs in the SVF are characterized by adherence to plastic and the expression of specific cell surface marker (e.g., CD73, CD90, CD105), confirming their identity. This protocol provides a method for isolating viable AD-MSCs for downstream applications.

Lipid droplets are cellular organelles that regulate various cellular process such as lipid and energy metabolism. The first morphological indication of adipogenesis is the accumulation of multiple small intracellular lipid droplets. Here, we describe a microscopy technique that can be used to quantify lipid droplets in adipocytes.

Advanced cell-based and gene therapy products emerged during the 1990s as new health product categories for treating and curing previously untreatable or incurable conditions. These products are complex, diverse and therapeutically specific, requiring specialised regulatory frameworks. During the last three decades, several jurisdictions have constructed specific regulatory frameworks to ensure these products’ safety, clinical efficacy and quality. As these are new and disruptive products, these frameworks are continuously evolving. However, South Africa (SA)’s regulatory frameworks for medicines, human biological materials and genetically modified organisms have not kept pace with scientific and technological developments, leaving regulatory gaps. We briefly describe these novel products and their regulatory frameworks, and propose a way forward in SA.

Neonatal encephalopathy suspected to be due to hypoxic ischaemic encephalopathy (NESHIE) carries the risk of death or severe disability (cognitive defects and cerebral palsy). Previous genetic studies on NESHIE have predominantly focused on exomes or targeted genes. The objective of this study was to identify genetic variants associated with moderate-severe NESHIE through whole-genome, unbiased analysis. Variant filtering and prioritization were performed, followed by association testing both on a case-control basis and to compare the grades of severity and/or progression. Association testing on neonates with NESHIE (N = 172) and ancestry-matched controls (N = 288) produced 71 significant genetic variants (false discovery rate corrected p-value < 6.2 × 10-4), all located in non-coding regions and not previously implicated in NESHIE. Disease-associated variants in non-coding regions are considered to affect regulatory functions, possibly by modifying gene expression, promoters, enhancers, or DNA structure. The most significant variant was at position 6:162010973 in the Parkin RBR E3 ubiquitin protein ligase (PRKN) intron. Intronic variants were also identified in genes involved in inflammatory processes (SLCO3A1), DNA repair (ZGRF1), synaptogenesis (CNTN5), haematopoiesis (ASXL2), and the transcriptional response to hypoxia (PADI4). Ten variants were associated with a higher severity or lack of improvement in NESHIE, including one in ADAMTS3, which encodes a procollagen amino protease with a role in angiogenesis and lymphangiogenesis. This analysis represents one of the first efforts to analyze whole-genome data to investigate the genetic complexity of NESHIE in diverse ethnolinguistic groups of African origin and provides direction for further study.

Osteogenesis, which refers to the formation of bone tissue, can be studied in vitro to elucidate the mechanisms of bone development, identify key factors and cytokines, and enhance potential regenerative medicine applications in tissue engineering and transplantation. Adipose-derived stromal/stem cells (ASCs) are particularly promising for these studies due to their capacity to differentiate into various mesodermal lineage cells, including osteoblasts. To induce osteogenic differentiation, ASCs are cultured in a complete growth medium (CGM) supplemented with β-glycerophosphate, ascorbate-2-phosphate and dexamethasone over a period of at least 3 weeks. Osteogenic differentiation can be assessed through the deposition of hydroxyapatite, a key component of mineralized bone matrix, and the activity of alkaline phosphatase, an enzyme crucial for matrix calcification. This study provides a protocol for differentiating ASCs into osteoblasts using osteogenic differentiation medium (ODM) and evaluating the differentiation using Alizarin Red S (ARS) staining and alkaline phosphatase (ALP) assays. The methodology includes the preparation of CGM and ODM, as well as the specific procedures for ARS staining and ALP activity assays. The ARS staining involves fixation of cells, staining, and quantification of hydroxyapatite deposition, while the ALP assay measures enzyme activity. Both assays are normalized to cell counts, determined through DAPI staining and microscopic analysis. The results of these assays are intended to provide an accurate evaluation of osteogenic differentiation efficiency in ASCs.

Wharton's jelly-derived mesenchymal stromal/stem cells (WJSCs) are growing in popularity as an option for cell-based therapies. These cells are found within the gelatinous matrix of the umbilical cord, referred to as Wharton's jelly. WJSCs can be isolated by enzymatic tissue digestion or by using the explant method. Here, we discuss the explant method for the isolation of WJSCs.

Adipose-derived stromal/stem cells (ASCs) have generated great interest both with regard to research and their potential applications in translational clinical sciences. ASCs are often required for use at a later time relative to the time at which they were harvested. This requires appropriate conditions and procedures for storage and retrieval of these cells as needed. This protocol describes cryopreservation and thawing of ASCs without compromising the viability of the stored cells.

Introduction:General movements assessment (GMA), including the Motor Optimality Score-Revised (MOS-R) and the Hammersmith Infant Neurological Examination (HINE), has been shown in different settings to predict cerebral palsy (CP) and delayed neurodevelopment with high accuracy. However, their combined predictive ability has not been fully evaluated in infants with presumed hypoxic-ischaemic encephalopathy (HIE). Objective:This study aimed to assess the predictive ability of combined GMA, MOS-R, and HINE at 3 months in term or near-term infants diagnosed with presumed HIE, for neurodevelopmental outcome at 18 months. Methods:A cohort of presumed HIE infants treated with therapeutic hypothermia (TH) underwent GMA, MOS-R, and HINE at 12-15 weeks, and neurodevelopmental assessments using the Bayley Scales of Infant and Toddler Development Third Edition (BSID-III) at 9-12 and at 18-24 months of age. Combined early assessments were analysed for their predictive ability across different domains on the BSID-III. Results:Twenty-four infants were included; 7 (29%) had both 12-month and 18-month BSID-III assessments, 12 (50%) were seen only at 12 months, and 5 (21%) only at 18 months. Two infants with absent fidgety movements (FMs) and poor motor repertoire were later diagnosed with CP or showed delays in two domains on the BSID-III assessment at 18 months. While most infants had some abnormality in the MOS-R categories, only absent FMs and abnormal finger variability showed some association with the 18-month BSID-III assessment on univariate analysis. Of the four infants classified as at risk for CP on the HINE at 3 months, two had some motor abnormalities at 18 months. Combining the GMA, MOS-R, and HINE had high sensitivity and negative predictive value (100%), but low specificity (0-17.6%) and positive predictive value (6.2%-25%) for the BSID-III outcome. Conclusion:Combining GMA, MOS-R, and HINE was highly sensitive in this cohort, but had low specificity. This may lead to overdiagnosis, but it may be a useful screening tool for identifying typically developing infants who do not need intensive follow-up.

Adipose-derived stromal/stem cells (ASCs) have raised a great deal of interest in research and regenerative medicine with potential applications in the treatment of several diseases. ASCs are often required in large numbers for use in research and clinical applications. Standardization of isolation methods, expansion, and characterization will ensure quality of the cells as well as safety in their use. This protocol describes the expansion and characterization of ASCs and highlights ASC viability assessment following expansion.

The ability to isolate adipose-derived stromal/stem cells (ASCs) with relative ease has generated a great deal of interest in their potential use for regenerative medicine purposes. ASCs have shown promise in areas such as graft-versus-host disease and wound healing. Murine ASCs (mASCs) can be isolated from various adipose tissue sites. To further explore their therapeutic potential, it is crucial to be able to successfully isolate, expand, and characterize these cells. White adipose tissue (WAT) and brown adipose tissue are the main adipose tissue categories. This chapter presents a protocol for the isolation, expansion, and characterization of mASCs from excised WAT.