Melissa H Little

University of Queensland, Brisbane, Queensland, Australia

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Publications (251)

  • Minoru Takasato · Pei X Er · Han S Chiu · Melissa H Little
    Article · Aug 2016 · Nature Protocols
  • Minoru Takasato · Melissa H. Little
    [Show abstract] [Hide abstract] ABSTRACT: Directed differentiation of human pluripotent stem cells (hPSCs) can provide us any required tissue/cell types by recapitulating the development in vitro. The kidney is one of the most challenging organs to generate from hPSCs as the kidney progenitors are composed of at least 4 different cell types, including nephron, collecting duct, endothelial and interstitium progenitors, that are developmentally distinguished populations. Although the actual developmental process of the kidney during human embryogenesis has not been clarified yet, studies using model animals accumulated knowledge about the origins of kidney progenitors. The implications of these findings for the directed differentiation of hPSCs into the kidney include the mechanism of the intermediate mesoderm specification and its patterning along with anteroposterior axis. Using this knowledge, we previously reported successful generation of hPSCs-derived kidney organoids that contained all renal components and modelled human kidney development in vitro. In this review, we explain the developmental basis of the strategy behind this differentiation protocol and compare strategies of studies that also recently reported the induction of kidney cells from hPSCs. We also discuss the characterisation of such kidney organoids and limitations and future applications of this technology.
    Article · Aug 2016
  • Melissa H. Little
    [Show abstract] [Hide abstract] ABSTRACT: The successful generation of kidney-like structures from human pluripotent stem cells, although slower to come than other tissue types, brings the hope of new therapies. While the demand for alternative treatments for kidney failure is acute, huge challenges remain to move these exciting but preliminary results toward clinical use.
    Article · Jun 2016 · Cell stem cell
  • Alexander N. Combes · James G. Lefevre · Sean Wilson · [...] · Melissa H. Little
    [Show abstract] [Hide abstract] ABSTRACT: Morphogenesis of the mammalian kidney requires reciprocal interactions between two cellular domains at the periphery of the developing organ: the tips of the epithelial ureteric tree and adjacent regions of cap mesenchyme. While the presence of the cap mesenchyme is essential for ureteric branching, how it is specifically maintained at the tips is unclear. Using ex vivo timelapse imaging we show that cells of the cap mesenchyme are highly motile. Individual cap mesenchyme cells move within and between cap domains. They also attach and detach from the ureteric tip across time. Timelapse tracks collected for >800 cells showed evidence that this movement was largely stochastic, with cell autonomous migration influenced by opposing attractive, repulsive and cell adhesion cues. The resulting swarming behaviour maintains a distinct cap mesenchyme domain while facilitating dynamic remodelling in response to underlying changes in the tip.
    Article · Jun 2016 · Developmental Biology
  • Melissa H. Little · Pamela Kairath
    [Show abstract] [Hide abstract] ABSTRACT: The treatment of renal failure has changed little in decades. Organ transplantation and dialysis continue to represent the only therapeutic options available. However, decades of fundamental research into the response of the kidney to acute injury and the processes driving progression to chronic kidney disease are beginning to open doors to new options. Similarly, continued investigations into the cellular and molecular basis of normal kidney development, together with major advances in stem cell biology, are now delivering options in regenerative medicine not possible as recently as a decade ago. In this review, we will discuss advances in regenerative medicine as it may be applied to the kidney. This will cover cellular therapies focused on ameliorating injury and improving repair as well as advancements in the generation of new renal tissue from stem/progenitor cells.
    Article · May 2016 · Kidney International
  • Minoru Takasato · Pei X Er · Han S Chiu · [...] · Melissa H Little
    Article · Apr 2016 · Nature
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    [Show abstract] [Hide abstract] ABSTRACT: The epicardium has a critical role during embryonic development, contributing epicardium-derived lineages to the heart, as well as providing regulatory and trophic signals necessary for myocardial development. Crim1 is a unique trans-membrane protein expressed by epicardial and epicardially-derived cells but its role in cardiogenesis is unknown. Using knockout mouse models, we observe that loss of Crim1 leads to congenital heart defects including epicardial defects and hypoplastic ventricular compact myocardium. Epicardium-restricted deletion of Crim1 results in increased epithelial-to-mesenchymal transition and invasion of the myocardium in vivo, and an increased migration of primary epicardial cells. Furthermore, Crim1 appears to be necessary for the proliferation of epicardium-derived cells (EPDCs) and for their subsequent differentiation into cardiac fibroblasts. It is also required for normal levels of cardiomyocyte proliferation and apoptosis, consistent with a role in regulating epicardium-derived trophic factors that act on the myocardium. Mechanistically, Crim1 may also modulate key developmentally expressed growth factors such as TGFβs, as changes in the downstream effectors phospho-SMAD2 and phospho-ERK1/2 are observed in the absence of Crim1. Collectively, our data demonstrates that Crim1 is essential for cell-autonomous and paracrine aspects of heart development.
    Full-text Article · Jan 2016 · Scientific Reports
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    Full-text Dataset · Jan 2016
  • Yu Leng Phua · Thierry Gilbert · Alexander Combes · [...] · Melissa H Little
    [Show abstract] [Hide abstract] ABSTRACT: Congenital medullary dysplasia with obstructive nephropathy is a common congenital disorder observed in paediatric patients and represents the foremost cause of renal failure. However, the molecular processes regulating normal papillary outgrowth during the postnatal period are unclear. In this study, transcriptional profiling of the renal medulla across postnatal development revealed enrichment of non-canonical Wnt signalling, vascular development and planar cell polarity genes, all of which may contribute to perinatal medulla / papilla maturation. These pathways were investigated in a model of papillary hypoplasia with functional obstruction, the Crim1(KST264) (/) (KST264) transgenic mice. Postnatal elongation of the renal papilla via convergent-extension was unaffected in the Crim1(KST264) (/) (KST264) hypoplastic renal papilla. In contrast, these mice displayed a disorganised papillary vascular network, tissue hypoxia and elevated Vegfa expression. In addition, we demonstrate the involvement of accompanying systemic hypoxia arising from placental insufficiency, in appropriate papillary maturation. In conclusion, this study highlights the requirement for normal vascular development in collecting duct patterning, development of appropriate nephron architecture and perinatal papillary maturation, such that disturbances contribute to obstructive nephropathy. {EdQ please check that your meaning has been retained} OK.
    Article · Jan 2016 · The Journal of Pathology
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    Minoru Takasato · Pei X. Er · Han S. Chiu · Melissa H. Little
    Full-text Article · Oct 2015
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    Minoru Takasato · Pei X Er · Han S Chiu · [...] · Melissa H Little
    [Show abstract] [Hide abstract] ABSTRACT: The human kidney contains up to 2 million epithelial nephrons responsible for blood filtration. Regenerating the kidney requires the induction of the more than 20 distinct cell types required for excretion and the regulation of pH, and electrolyte and fluid balance. We have previously described the simultaneous induction of progenitors for both collecting duct and nephrons via the directed differentiation of human pluripotent stem cells. Paradoxically, although both are of intermediate mesoderm in origin, collecting duct and nephrons have distinct temporospatial origins. Here we identify the developmental mechanism regulating the preferential induction of collecting duct versus kidney mesenchyme progenitors. Using this knowledge, we have generated kidney organoids that contain nephrons associated with a collecting duct network surrounded by renal interstitium and endothelial cells. Within these organoids, individual nephrons segment into distal and proximal tubules, early loops of Henle, and glomeruli containing podocytes elaborating foot processes and undergoing vascularization. When transcription profiles of kidney organoids were compared to human fetal tissues, they showed highest congruence with first trimester human kidney. Furthermore, the proximal tubules endocytose dextran and differentially apoptose in response to cisplatin, a nephrotoxicant. Such kidney organoids represent powerful models of the human organ for future applications, including nephrotoxicity screening, disease modelling and as a source of cells for therapy.
    Full-text Article · Oct 2015 · Nature
  • Andrew Mallett · Barbara Maier · Pei Xuan Er · [...] · Melissa Little
    [Show abstract] [Hide abstract] ABSTRACT: Background: The reprogramming of somatic cells into iPSCs provides potential to model human diseases in vitro, as has been demonstrated in the cardiac and neuronal fields. Nephrology has not yet benefited from these advancements primarily due to lack of a robust kidney differentiation protocol. Methods: We aimed to generate and characterize iPSCs from patients with different GRDs. Families with clinically diagnosed GRD were recruited from a Renal Genetics Clinic for concurrent research-based massively parallel sequencing (MPS) and iPSC generation. One affected and unaffected member of each family was recruited. To establish transgene-free iPSC lines, fibroblasts were isolated via skin biopsy and reprogrammed using non-integrating Sendai virus (21day protocol). Results: 7 families (14 participants) were recruited with a variety of GRD diagnoses. Fibroblast culture was successful in 6 families. Established iPSC lines have typical hESC-like morphology and express pluripotency markers after 4 (TRA1-60) and 15 passages (NANOG). Moreover, iPCS lines have cleared the Sendai virus vectors, as confirmed by RT PCR after only 7 passages. G-band analysis of 2 lines from each isolation confirmed that each of the derived lines had maintained the normal karyotype after reprogramming. Paired patient and control iPSC are being redifferentiated towards kidney employing an established protocol. Renal organoids will be analyzed using IF, FACS and transcriptional profiling. Conclusions: Patient derived iPSC have been generated and renal redifferentiation commenced. We hope to uncover the biological consequences of novel genetic variants causing GRD as identified via MPS, thereby beginning to explain patient phenotypes and disease pathogenesis.
    Conference Paper · Oct 2015
  • [Show abstract] [Hide abstract] ABSTRACT: Background: Genetic Renal Disease (GRD) accounts for 10% of adults and 50% of children with end stage kidney disease. Advances in molecular genetic diagnostic testing and multidisciplinary renal clinic models suggest opportunities for clinical redesign. Here we describe the initial characteristics, experience and outcomes of the first Australian multidisciplinary RGC. Methods: A retrospective audit was undertaken of the RBWH Adult Conjoint RGC (1st year of operation; Aug’13-Jul’14). Each encounter involved a nephrologist, clinical geneticist and genetic counselor. Results: 27/48 patients (56%) encountered had a known GRD, 9/48 (19%) had a suspected GRD and 12/48 (25%) had an unknown GRD with positive family history (FHx). Patients were most commonly referred by nephrologists (66%).The most common GRD category & diagnosis were cystic kidney disease (49%) and Autosomal Dominant Tubulointerstitial Kidney Disease (25%). Extra-renal features were associated with GRD in 26/48 (54%). During consultations differential diagnoses were explored (54%), management advice provided (83%) and genetic counseling undertaken (79%). A genetic test was requested in 58.3%, most commonly being indicated for combined diagnostic and genetic counseling reasons (78.6%). Of 12 returned genetic test results, 7 were positive and 1 was a variant of uncertain significance. Two negative results have prompted additional genetic testing. Of 27 patients referred with a known GRD, 6 now have a new suspected GRD. The majority of those referred with either a suspected (6/9) or unknown GRD diagnosis (10/12) now have a new suspected or confirmed GRD. Conclusions: These findings demonstrate the early successful operation of this RGC model including diagnostic and genetic counseling benefit. Future assessment of clinical outcomes and expansion to telehealth and paediatric settings is anticipated.
    Conference Paper · Oct 2015
  • Melissa H. Little
    [Show abstract] [Hide abstract] ABSTRACT: Although we know that mesenchymal progenitors give rise to nephrons in the kidney, how they balance self-renewal versus differentiation is still unclear. In this issue of Developmental Cell, Chen et al. (2015) show that nephron progenitors age, but not necessarily irreversibly: old progenitors can be "rejuvenated" by a young crowd. Although we know that mesenchymal progenitors give rise to nephrons in the kidney, how they balance self-renewal versus differentiation is still unclear. In this issue of Developmental Cell, Chen et al. (2015) show that nephron progenitors age, but not necessarily irreversibly: old progenitors can be "rejuvenated" by a young crowd.
    Article · Oct 2015 · Developmental Cell
  • [Show abstract] [Hide abstract] ABSTRACT: Background: GRD accounts for 10% of adults and 50% of children with end stage kidney disease. An unknown proportion of cases remain refractory to current clinical genetic tests. Advances in MPS enable attempts to address this in a research setting. We aimed to utilize MPS with pedigree analysis in unresolved cases of GRD. Methods: Families with clinically diagnosed GRD were recruited in whom clinical genetic testing was either negative or not available. Whole exome sequencing was undertaken and analyzed with custom in house bioinformatics assessment tools accounting for all inheritance patterns. Identified variants of interest were confirmed using Sanger sequencing in clinical laboratories. Results: 14 families (62 participants) were recruited with a variety of GRD diagnoses and modes of inheritance. A molecular genetic diagnosis has been resolved in 5 families. These include mutations in RMND1 (compound heterozygous), IFT140 (compound heterozygous), HNF4A (heterozygous), COL4A5 (hemizygous) and tRNA(Phe) (mtDNA) genes. All have been confirmed in a clinical diagnostic laboratory and reported back to the participants with further genetic counseling. Research continues in the remaining 9 families, including application of whole genome sequencing. No reportable incidental genetic findings were identified. Conclusions: MPS confirmed and clarified a clinical genetic diagnosis in 5/14 families with previously and diagnostically refractory GRD. These results were integrated into clinical practice and demonstrate an emerging role for MPS in the delivery of clinical care. Further studies are required to resolve the remaining families.
    Conference Paper · Oct 2015
  • [Show abstract] [Hide abstract] ABSTRACT: Aim: To investigate the genetic aetiology of deafness, lactic acidosis (LA) and nephropathy in Australian patients. Background: RMND1 is an integral inner membrane mitochondrial protein. Mutations in RMND1 cause an autosomal recessive mitochondrial oxidative phosphorylation deficiency disorder recently phenotypically expanded to encompass syndromic nephropathy. Methods: Two Australian patients with global developmental delay (GDD), deafness, LA and nephropathy underwent whole exomic sequencing (WES). Results: Patient 1 presented in the neonatal period with seizures and sensorineural hearing loss (SNHL) having been born at 24 weeks gestation. The child had tubulopathy with polyuria, hyponatraemia, hyperkalaemia, metabolic acidosis (LA), positive sweat test (negative CFTR analysis) and progressive renal impairment. Renal ultrasound showed normal sized echogenic kidneys. Renal biopsy demonstrated chronic tubulointerstitial nephritis and nephronophthisis. WES identified compound heterozygous RMND1 mutations (c.[713A>G][829_830del];p.[ Asn238Ser][ Glu277Glyfs*20]). At 4years, GDD continues to improve, bilateral cochlear implants have been successful and renal impairment continues to progress (eGFR 33 ml/min/1.73 m2). Patient 2 was born at term by normal delivery and had unremarkable neonatal course except for bilateral SNHL. At 5 months of age, he was noted to have generalised hypotonia, poor weight gain, GDD, LA and 3-methylglutaconic aciduria in the context of a viral illness. A homozygous RMND1 mutation (c.[1349G>C];p.[*450Serext*32]) was identified by WES (Melbourne Genomics Health Alliance). Subsequent screening for renal disease showed hyperkalemia and renal hypodysplasia. At 18 months, he is globally delayed and hypotonic. Conclusions: These cases reinforce the relationship between RMND1 mutations and autosomal recessive nephropathy associated with SNHL and LA, whilst demonstrating the utility of WES in diagnosis. Tubulointersitital changes on renal biopsy suggest a potential relationship with other forms of ciliopathy. This and how oxidative phosphorylation deficiencies mediate renal disease require further investigation.
    Conference Paper · Sep 2015
  • [Show abstract] [Hide abstract] ABSTRACT: Aim: To identify the genetic cause of genetic renal disease (GRD) in a large Australian family whose pedigree is consistent with autosomal dominant or mitochondrial inheritance. Background: Mutations in tRNA(Phe) are rarely reported to cause renal disease, usually with syndromic features such as epilepsy and myopathy. Methods: A large Australian family with GRD underwent whole exomic sequencing (WES) with mtGenome capture, and mtDNA sequencing. Results: The family has extensive kidney disease historically identified as “MedullaryCystic Kidney Disease”. Six surviving members receive renal replacement therapy or have a functioning renal transplant. Seven further deceased family members had Chronic or End-Stage Kidney Disease. No clear history of an extrarenal phenotype is apparent, though one affected family member has an undiagnosed oromotor dysphagia parkinsonian syndrome and another died of apparent herpetic encephalitis. Renal histopathology in three affected family members >30 years ago demonstrated tubulointerstitial disease with mild tubular dilatation and some corticomedullary cyst formation. WES did not reveal any candidate variants in the nuclear genome. mtDNA sequencing identified the T616C mutation in the anticodon loop of tRNA(Phe). This mutation was homoplasmic in blood and present in all affected family members including 4th degree cousins. Northern blot demonstrated decreased phenylalanine tRNA in affected fibroblasts compared to wild-type control fibroblasts. A respiratory chain defect was present in affected fibroblasts and cybrids compared to wild-type, confirming the mitochondrial origin. Conclusions: Here we confirm the T616C tRNA(Phe) mutation as causative for Mitochondrially Inherited Tubulointerstitial Kidney Disease, in the apparent absence of extrarenal syndromic features. This has implications for genetic counselling and demonstrates the utility of collaborative genetic diagnostic approaches. The pathobiology of how such mitochondrial mutations cause renal disease requires further research.
    Conference Paper · Sep 2015
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    Andrew Mallett · Chirag Patel · Barbara Maier · [...] · Melissa Little
    [Show abstract] [Hide abstract] ABSTRACT: BACKGROUND: Genetic renal diseases (GRD) are a heterogeneous and incompletely understood group of disorders accounting for approximately 10 % of those diagnosed with kidney disease. The advent of Next Generation sequencing and new approaches to disease modelling may allow the identification and validation of novel genetic variants in patients with previously incompletely explained or understood GRD. METHODS/DESIGN: This study will recruit participants in families/trios from a multidisciplinary sub-specialty Renal Genetics Clinic where known genetic causes of GRD have been excluded or where genetic testing is not available. After informed patient consent, whole exome and/or genome sequencing will be performed with bioinformatics analysis undertaken using a customised variant assessment tool. A rigorous process for participant data management will be undertaken. Novel genetic findings will be validated using patient-derived induced pluripotent stem cells via differentiation to renal and relevant extra-renal tissue phenotypes in vitro. A process for managing the risk of incidental findings and the return of study results to participants has been developed. DISCUSSION: This investigator-initiated approach brings together experts in nephrology, clinical and molecular genetics, pathology and developmental biology to discover and validate novel genetic causes for patients in Australia affected by GRD without a known genetic aetiology or pathobiology.
    Full-text Article · Sep 2015 · BMC Nephrology
  • [Show abstract] [Hide abstract] ABSTRACT: Aim: To identify a rare genetic cause for atypical autosomal dominant Fanconi renotubulopathy mimicking Dent’s Disease amongst Australian patients. Background: The HNF4A gene is associated with “Maturity Onset Diabetes of the Young Type 1” however a specific variant has recently been identified causing distinctive autosomal dominant renal tubulopathy. Translation to clinical diagnostic sequencing is desirable. Methods: Two Australian patients with atypical Fanconi renotubulopathy respectively underwent small-pedigree whole exomic sequencing (WES) and diagnostic clinical exome sequencing via the Australian Renal Genetic Disorders Panels (ARGP) at Westmead. Results: Patient 1 presented at 10 years with rickets (requiring multi-level orthopaedic procedures), short stature, asymptomatic low-molecular-weight proteinuria with Fanconi renotubulopathy and progressive chronic kidney disease (CKD). Development was otherwise normal with past history of surgically repaired ventricular septal defect at 5 years. Growth hormone therapy precipitated diabetes mellitus, which resolved upon its cessation. Renal biopsy, imaging and CLCN5/OCRL were normal. Small-pedigree WES identified the de novo p.R76W (NM_000457.4): c.[187C>T];[=]) variant in HNF4A. Patient 2 presented in early childhood with asymptomatic low-molecular-weight proteinuria. She progressively developed Fanconi renotubulopathy, progressive CKD, recurrent morning ketotic hypoglycaemia, hyperaldosteronism, minor osteopenia, subclinical rickets, intermittently raised intraocular pressure, hypermetropia, mild intellectual impairment, generalised joint hypermobility, and pancreatic hyperechogenicity of unknown cause. Renal biopsy, imaging and CLCN5 were normal. ARGP analysis was initially negative, however reanalysis based on recent reports of patients with the p.R76W variant in HNF4A, identified this variant (NM_000457.4):c.[187C>T][=]). Conclusions: These cases reconfirm the association of this heterozygous variant in HNF4A with atypical Fanconi renotubulopathy whilst demonstrating diagnostic translation and integration into the clinical ARGP service for identification of further cases. Further research into the pathogenic mechanism of this variant is required.
    Conference Paper · Sep 2015
  • M. H. Little · J. M. Vanslambrouck · M. Takasato · [...] · H. Chiu
    Conference Paper · Aug 2015

Publication Stats

6k Citations

Institutions

  • 2015
    • University of Queensland
      • Institute for Molecular Bioscience
      Brisbane, Queensland, Australia
    • The Royal Children's Hospital
      Melbourne, Victoria, Australia
  • 2011
    • University of Vic
      Vic, Catalonia, Spain
  • 2006
    • Monash University (Australia)
      • Monash Immunology and Stem Cell Laboratories (MISCL)
      Melbourne, Victoria, Australia
  • 2004
    • Nara Institute of Science and Technology
      Ikuma, Nara, Japan
  • 1998
    • Western General Hospital
      Edinburgh, Scotland, United Kingdom