Browsing by Author "Ballo, Robea"

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    Open Access
    Generating hair follicle inductive dermal papillae cells from adipose derived mesenchymal stem cells
    (2018) Brown, Alice Clare; Kidson, Sue; Ballo, Robea
    Current management options for cutaneous burn wounds, including split thickness skin grafts and cultured epithelial autografts, generate an epithelial barrier which lacks a dermal layer and skin adnexae including hair follicles and sebaceous glands. This results in a loss of pliability and contractures that cause functional and cosmetic impairment. Embryological hair follicle morphogenesis results from a complex series of mesenchymal-epithelial interactions and to date a method of generating de novo folliculogenesis from human cells has yet to be accomplished. Existing models rely on combining 'inductive’ dermal and 'receptive’ epithelial components and placing them within a suitable model. Epithelial cells are easily obtainable from skin biopsies therefore obtaining sufficient quantities of 'trichogenic’ dermal cells remains the most significant challenge of this approach. The main aim of this project is to contribute to the achievement of de novo folliculogenesis by generating dermal papillae (DP) like-spheroids using adipose derived mesenchymal stem cells (ASCs) that, when combined with responsive epithelial cells, would be capable of inducing hair follicle formation. ASCs were directed towards a hair follicle DP-like fate by culture using the hanging drop method and exposure to Wnt, mimicking signalling and mesenchymal condensation in embryological hair follicle induction. Gene expression analysis using RT-PCR showed that the DP-cell marker Versican is expressed at high levels in ASCs under routine culture conditions and the exposure of ASCs to Wnt results in a more than threefold increase in this expression. These results suggest that Wnt/β-catenin signalling may regulate DP cell aggregative growth through modifying versican expression possibly through binding of β-catenin to the TCF transcription factor complex. Culture of ASCs using the hanging drop method produces spheroids similar in size to human hair follicle DP. Histology of these spheroids demonstrates viable cells that flatten around the outside. The spheroids grow out when replated onto Matrigel in a 3D culture model and exhibit a morphology similar to that of primary hair follicle DP cells. Analysis of mRNA expression demonstrates that Versican expression is significantly upregulated in DP-like spheroids in the absence or presence of Wnt demonstrating that Versican may be responsible for both induction and maintenance of mesenchymal cell condensates. Alpha smooth muscle actin is expressed in low levels in ASC spheroids compared to ASCs in a monolayer and this may reflect a 'migratory’ myofibroblast like phenotype of ASCs in a monolayer similar to cells with the hair follicle dermal sheath. The addition of Wnt to ASC spheroids has no additional effect on Versican expression possibly reflecting a negative feedback loop resulting from high local concentrations of endogenous Wnt expression from ASCs. The results of this study show that spheroid cell culture and exposure to Wnt of ASCs results in cell clusters with similar morphology and gene expression to hair follicle DP cells. The novel method of DP-like cell generation described in this study makes use of cells that are readily obtainable from patients and require minimal time and manipulation in culture and therefore could potentially be rapidly translatable to clinical trials.
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    Induction of hair follicles using neonatal mouse dermis and human keratinocytes: relevance for improved burn wound treatments
    (2020) Malise, Thudzelani Takalani Austin; Kidson, Sue; Ballo, Robea
    Second degree burns result in the destruction of the skin as well as its adnexa. Following such burns the wound heals without the formation of skin appendages and hair follicles. In normal embryonic development hair follicle formation requires the interaction between epithelial (keratinocytes) and mesenchyme cells. Attempts to recapitulate the process of hair induction in wounded skin in vitrousing human cells have to date been unsuccessful. The aim of this project is to attempt to elicit the early steps of hair follicle formation (induction) by co-culturing primary human keratinocytes with embryonic murine mesenchyme cells and assessing changes in expression patterns of genes associated with or reflective of induction. Mesenchymal cells and keratinocytes cells were obtained by enzymatically digesting dorsal neonatal mouse skin and neonatal human foreskin using dispase and collagenase. Cells were cultured separately, and their growth dynamics measured. The isolated neonatal mouse mesenchymal cells were shown to have hair induction potential because they expressed dermal papilla signature genes, Alp, Sox2 and Vcan. However, this characteristic was lost during in vitro propagation suggesting that mesenchymal cells lose their hair inductive potential during culture. In contrast, when cultured at high densities or in spheroids in hanging drops, the dermal papilla signature genes were upregulated suggesting that this might be a way to maintain inductive potential. Primary foreskin keratinocytes expressed high levels of basal layer marker, keratin 5 (K5), and low levels of the early differentiation marker, K10, suggesting that the isolated keratinocytes have stem cell properties. When co-cultured with neonatal mouse mesenchymal cells using Transwells, the mesenchymal cells were able to elicit colony formation on keratinocytes in co-cultures, indicating that they support keratinocyte proliferation. It was not possible to do hair follicle induction marker analysis of human foreskin keratinocytes cocultures because of challenges and difficulties encountered during expansion. Therefore, Immortalised HaCaT keratinocytes were tested. HaCaT keratinocytes were shown to be induced during cocultures because they upregulated Wnt signalling genes, β-catenin and NF-KB. As an additional approach, human foreskin keratinocytes were cultured in medium containing Wnt signalling pathway ligand, Wnt3a. β-catenin and NF-KB were slightly reduced, and only Lef1 was upregulated in human foreskin keratinocytes cultured in Wnt3a conditioned medium. The results of this study show that neonatal mouse mesenchymal cells have hair inducing capabilities and it is lost by in vitro propagation and can be restored by spheroid cell culture. The results also demonstrate that human foreskin keratinocytes need to be expanded using efficient culture methods to maintain an undifferentiated state.
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    Investigating the role of a FAM111B mutation in hereditary fibrosing poikiloderma (POIKTMP) using induced pluripotent stem cell (iPSC) model
    (2019) Gumede, Dimakatso B; Kidson, Sue H; Ballo, Robea; Mayosi, Bongani M
    Hereditary fibrosing poikiloderma is an autosomal dominant disorder that is characterised by mottled pigmentation and telangiectasia, accompanied by tendon contractures, myopathy and pulmonary fibrosis (POIKTMP). Mutations in POIKTMP cases have been shown to harbour the Family with sequence similarity 111B (FAM111B) gene. However, its function is unknown. The aim of this study was to investigate the causative role of the FAM111B mutation (c.1861T>G) in the multi-systemic fibrosis affecting the South African kindred with POIKTMP. Dermal fibroblasts from two affected siblings and a familial control were reprogrammed into induced pluripotent stem cells (iPSCs) via the Sendai virus vector (SeVdp) packaged with pluripotency transgenes (OCT4; SOX2; KLF4; C-MYC). The derived iPSCs successfully showed a) endogenous expression of pluripotency markers (OCT4; NANOG; TRA-1-60), b) in vitro differentiation into the three germ layers (endoderm; mesoderm; ectoderm) and c) normal karyotyping. Next, the iPSCs from two patients, a Familial control and a Non-familial control were differentiated into mesenchymal stem/stromal cells (iPSC-MSCs) as a cell model in this study. Characterisation of derived iPSC-MSCs indicated positive expression of MSC markers (CD73; CD90; α-SMA). Differentiation of iPSC-MSCs demonstrated adequate osteogenicity but limited adipogenicity. Patient-derived iPSC-MSCs were thereafter analysed by qPCR and collagen staining to determine whether the FAM111B mutation alters endogenous expression of pro-fibrotic markers as well as collagen synthesis in patient cells compared to controls. Messenger RNA expression of pro-fibrotic markers (COL1A1; COL3A1; α-SMA) was similar between patient and control iPSC-MSCs. Collagen staining and quantification also showed no statistical differences between patient and control cells. These results suggest that FAM111B does not directly alter the expression of these profibrotic genes in this in vitro model system. Growth curves were then carried out to investigate if the FAM111B mutation modulates cell proliferation and it was found that patient cells proliferated at a higher rate compared to controls. To explore the mechanisms underlying the rate change, analyses of FAM111B expression during cell cycle progressions were conducted. Extensive optimization experiments using the double thymidine block approach were necessary to establish the appropriate synchronization protocol, keeping in mind the extended doubling time of iPSCMSCs. The results revealed that FAM111B mRNA expression was temporally regulated, with a peak at the S-phase and low at the G2/M phase. While there were no pattern differences between patient and control cells, FAM111B mRNA expression was significantly higher in the patient cells compared to controls at the G1- and S-phase. These results suggest that the mutation in FAM111B might affect the stability or perdurance of the mRNA. Unfortunately, analysis of the FAM111B protein data was inconclusive. Problems related to synchronization of the cells and the specificity of the antibody would have to be rectified in order to follow this further. The overall findings in this in vitro study reveal that the FAM111B mutation does not alter expression of pro-fibrotic markers but does affect the cell proliferation rate of patient cells compared to controls. Future work will focus on further optimisation of iPSC-MSCs synchronisation to determine correlation of FAM111B mRNA and protein expression during cell cycle progression in the patient cells. Furthermore, 3D in vitro cellular models that recapitulate some parts of the POIKTMP phenotype will need to be created. Future work will also explore the gain-of-function hypothesis to further understand the role of FAM111B in fibrosis and cancer phenotype in POIKTMP.
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    An investigation into the molecular basis of familial forms of osteoarthropathy in South Africa
    (1998) Ballo, Robea; Beighton, Peter; Ramesar, Rajkumar
    Generalised osteoarthritis (OA) is a common disorder of the joints which can lead to pain and disability. Identification of the determinant gene(s) is limited in part by the lack of Mendelian inheritance in most forms of the disorder, the combination of genetic and environmental influences and the late development of the condition. An approach to the investigation of the aetiology of OA would be to take advantage of the monogenic basis of inherited skeletal dysplasias in which OA is a major component. For this reason, the molecular genetic basis of the epiphyseal dysplasias, which encompass a spectrum of phenotypes ranging from mild to severe skeletal involvement, is addressed in this thesis. Familial skeletal disorders in South Africa in which OA is a major feature were identified and investigated using intragenic and closely linked microsatellite markers in order to determine linkage to candidate genes. Mutational analysis was undertaken to identify the genetic defect.
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    Investigation of the mechanisms underlying the effects of hyperglycaemia on cardiac structural and electrical remodelling
    (2022) Aboalgasm, Hamida; Gwanyanya, Asfree; Ballo, Robea
    Background: Diabetes mellitus with uncontrolled hyperglycaemia is a major cause of cardiovascular complications and mortality. The developing foetal heart in-utero is particularly susceptible to hyperglycaemia through pathological remodelling, which results in life-long structural abnormalities such as cardiomyopathy and electrical defects like arrhythmias. However, the underlying mechanisms and potential therapeutic drug targets remain unclear. In this study, a cardiac developmental cellular model was used to study hyperglycaemia-induced remodelling. Methods: Mouse embryonic stem cells (mESCs) were differentiated into pulsatile, cardiac-like cells via embryoid body (EB) formation and cultured under baseline- or high glucose conditions. A Ca2+ -sensitive fluorescent dye Fluo-4 was used to measure calcium transients and a voltage-sensitive dye di-4-ANEPPS was used to record action potentials. Cellular biomarkers were detected using immunocytochemistry, confocal microscopy, and Western blotting as well as terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) and 5-ethynyl-2-deoxyuridine (EdU) assay. Results: Undifferentiated mESCs were positive for pluripotent transcription factors Nanog and Oct3/4, whereas the cardiac differentiated mESCs were positive for cardiac proteins troponin T, α-actinin 2, connexin 43, sarco-endoplasmic reticulum calcium ATPase 2 (SERCA 2) and α- and β-myosin heavy chain. Hyperglycaemia decreased the number of beating EBs, their beating rate, and their amplitude of contraction. It also decreased the calcium transient amplitude and the contractile response to ryanodine receptor stimulation by caffeine but did not alter the SERCA 2 expression. The amplitude and duration of action potentials in beating EBs were not altered by hyperglycaemia. However, structural changes included a decrease in EB size and expression of myofilament proteins, α-actinin and α- and β-myosin heavy chain and a disruption of the striated organization of the myofilaments. Hyperglycaemia increased the proportion of TUNEL-positive cells and the expression of the pro-apoptotic marker cytochrome c and decreased the anti-apoptotic protein Bcell lymphoma 2 but did not alter the mitochondrial staining with Mitotracker. It also increased the oxidative stress marker nitrotyrosine but did not alter the extent of EdU nuclear staining nor the expression of the receptor of advanced glycation end-product. The antioxidant n-acetyl cysteine decreased the fraction of hyperglycaemia-induced TUNEL-positive cells and improved the α-actinin striated pattern. Conclusion: Hyperglycaemia suppressed the cardiac differentiation and contractile activity of mESCs as well as disrupted the cardiac myofilament organisation and expression. These effects of hyperglycaemia were likely mediated by mitochondrial-dependent apoptosis triggered by oxidative stress as well as by the abnormalities in calcium signalling. These results have potential clinical implications in foetal diabetic cardiac disease and add novel insights into the mechanistic factors that represent new therapeutic drug targets in the developing foetal heart.
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    Molecular analysis of decay accelerating factor as a potential susceptibility factor to developing treatment resistant extraocular muscle involvement in Myasthenia Gravis
    (2009) Uwimpuhwe, Henriette; Heckmann, Jeannine; Ballo, Robea; Prince, Sharon
    Myasthenia gravis (MG) is an autoimmune disorder in which auto-antibodies directed at the acetylcholine receptors (AChR) of the neuromuscular junction (NMJ) block, alter or destroy their targets. The anti-AChR antibodies cause activation of the classical complement pathway leading to inflammatory injury at the NMJ. Decay Accelerating Factor (DAF), a member of complement regulatory proteins, prevents activation of autologous components of complement pathways. The absence of DAF, in knock-out mouse models, has been shown to significantly increase the susceptibility to experimental autoimmune MG. A previous study showed that a high proportion of South African MG patients of African genetic ancestry develop immunosuppressive therapyresistant extraocular muscle (EOM) dysfunction. We hypothesized that these patients have deficient DAF expression in their EOMs resulting in less protection from complement injury.
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    Socio-ecological risk factors, explanatory models and treatment-seeking behaviours associated with Mseleni joint disease: a biocultural mixed methods study
    (2022) Dinkele, Elizabeth Sarah; Gibbon, Victoria E; Ballo, Robea
    Mseleni Joint Disease (MJD) is a crippling osteoarthropathy of unknown aetiology endemic to southern African Bantu-language speakers in a remote region of Northern KwaZulu-Natal, South Africa. Effective management of MJD has been hindered by limited insight into risk factors, explanatory models or treatment-seeking behaviours in those affected. Until MJD is better understood, disability, unemployment and dependence on social assistance grants and family income for subsistence will remain a reality for those affected. A mixed methods study was conducted with the aims of examining risk factors, explanatory models and treatment-seeking behaviours associated with MJD. The distribution, differential diagnosis and treatment of MJD were statistically analysed using medical records (n=723), MJD-patient surveys (n=37) and a meta-analysis. Socio-economic and cultural risk factors were assessed from surveys (n=99) and census publications. Interviews with MJD patients (n=6), nurses (n=7) and doctors (n=9) were qualitatively analysed for themes pertaining to perceptions, experiences and treatment-seeking for MJD. A point prevalence of 9% was estimated. Women were nearly twice as likely to have MJD than men (OR= 1.89; p=0.03) and the likelihood of MJD increased almost three-fold in those older than 50 years (OR= 2.83; p<0.01). Age was a confounder of the association between gender and MJD, as the sample was skewed in the representation of elderly women. MJD was only detected in patients older than 35 years, indicative of a later onset age than previously reported. The prevalence of MJD in settlements along tar and concrete roads, with access to public transport but limited piped water was suggestive of environmental risk factors or differential access to hospital-based care. Explanatory models of MJD were supernatural (witchcraft or ancestral displeasure); natural (nutritional deficiencies, 'genetics' and/or environmental); and/or social (gender-based practices and lifestyle). MJD patients described supernatural and natural aetiologies, and conceptualised disability as an inevitable reality. Consequently, patients reported taking few measures to prevent joint immobility, focussing instead on immediate symptomatic relief. Psychosocial and systemic barriers to treatment were suggestive of a disconnect between traditional African healing and Western biomedicine. This work demonstrates the value of the biocultural approach in identifying spatial, ecological, social and cultural processes that shape population patterns of health and disease.
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    Treatment-resistant ophthalmoplegia in Myasthenia gravis: extraocular muscle pathology, the role of TGFβ1 and the derivation of induced pluripotency towards 'disease-in-a-dish' modeling
    (2016) Rautenbach, Robyn Marié; Heckmann, Jeannine M; Ballo, Robea
    Myasthenia gravis (MG) is an autoimmune disease in which pathogenic antibodies target specific neuromuscular junction proteins, most frequently acetylcholine receptors (AChR). Among those without detectable AChR-antibodies, a subgroup of patients has antibodies directed against muscle-specific tyrosine kinase (MuSK). In MG the pathogenic antibodies result in failure of neuromuscular transmission with consequent fatiguable skeletal muscle weakness. MG frequently affects the extraocular muscles (EOMs) early in the course of the disease, resulting in diplopia and ptosis, which is usually reversible with treatment. A treatment-resistant ophthalmoplegia and ptosis occurs as a complication of MG in a distinct subset of cases referred to as OP-MG. The EOMs are highly specialised muscle tissue with a unique physiological and immunological microenvironment with a large satellite cell niche, a distinct muscle fibroblast population, different transcriptional and cellular signaling pathways and fewer intrinsic complement regulatory proteins to protect them against antibody- activated complement-mediated damage. We hypothesised that in OP-MG, there is a differential response of the EOMs to the underlying MG disease process(es) on a genetic and molecular level, resulting in abnormal myofibre homeostasis. We aimed to report descriptive clinical-pathological data pertaining to EOM function and histopathological and ultrastructural EOM tissue analysis of a patient with OP- MG versus that of a non-MG control (both consented to EOM donation at ocular realignment surgery). EOM tissue from an OP-MG individual with AChR- and MuSK- antibody negative MG, demonstrated predominantly myopathic pathology and ultrastructural evidence of mitochondrial stress. The OP-MG EOM findings differ from the control EOM, which showed normal muscle histopathology in a patient undergoing strabismus surgery for a sensory exotropia in a non-seeing eye (loss of retinal stimulus for fusion) and a similar duration of deviation. These OP-MG findings appear to better correlate with previously reported histology/ultrastructure in limb muscle in MuSK-positive MG rather than AChR-positive MG. We next focussed on transforming growth factor beta-1 (TGFβ1) as a critical cytokine involved in muscle repair. An auto-induction pathway in muscle allows TGFβ1 expression to influence the transdifferentiation of satellite cells into myofibroblasts or myoblasts. In orbital fibroblasts, TGFβ1 has also been shown to upregulate decay accelerating factor (DAF), a complement regulatory protein expressed at lower levels in EOMs than other muscles, which should protect against complement-mediated injury. We established OP-MG and control-MG phenotype-specific dermal fibroblast cell lines and performed immunoblotting to evaluate TGFβ1-induced Smad3 phosphorylation and Daf expression in mouse myotubes. We demonstrated repression of phosphorylated-Smad3, a marker of the canonical TGFβ1 pathway, in OP-MG versus control MG fibroblasts after treatment with TGFβ1. We also demonstrated that TGFβ1 significantly upregulates Daf expression levels in mouse myoblasts. Taken together, these results suggest that OP-MG fibroblasts (and possibly myofibroblasts) are likely to be more susceptible to complement-mediated damage and abnormal myofibrogenesis due to their altered response to TGFβ1 stimulation and secondary DAF upregulation. Finally we investigated the feasability of establishing an in vitro disease model for MG or OP-MG by reprogramming dermal fibroblasts into disease phenotype-specific induced pluripotent stem (iPS) cells. We successfully generated and characterised iPS cells for one individual. However, this process was very labour-intensive, cost-inefficient and time-consuming, taking approximately four months to establish pluripotency in a single patient and thereby limited its further application(s). In conclusion, the EOM ultrastructural findings of an OP-MG case are novel and show similar findings to those described in limb skeletal muscle of MuSK-positive MG patients. The TGFβ1 pathway appears to be differentially regulated in OP-MG compared to control-MG cases and this may impact DAF upregulation in the EOMs in MG patients. Finally, our group is exploring an alternative method of establishing a 'disease-in-a-dish' model that is more cost-effective and practically feasible than the iPS cell route.