Papers by Luis Villa-Diaz
Stem Cells
Insight into the molecular mechanisms governing the development and maintenance of pluripotency i... more Insight into the molecular mechanisms governing the development and maintenance of pluripotency is important for understanding early development and the use of stem cells in regenerative medicine. We demonstrate the selective inhibition of mTORC1 signaling is important for developing the inner cell mass (ICM) and the self-renewal of human embryonic stem cells. S6K suppressed the expression and function of pluripotency-related transcription factors (PTFs) OCT4, SOX2, and KLF4 through phosphorylation and ubiquitin proteasome-mediated protein degradation, indicating that S6K inhibition is required for pluripotency. PTFs inhibited mTOR signaling. The phosphorylation of S6 was decreased in PTF-positive cells of the ICM in embryos. Activation of mTORC1 signaling blocked ICM formation and the selective inhibition of S6K by rapamycin increased the ICM size in mouse blastocysts. Thus, selective inhibition of mTORC1 signaling supports the development and maintenance of pluripotency.
Stem Cells and Development, May 1, 2009
Human embryonic stem cell (hESC) culture is routinely performed using inactivated mouse embryonic... more Human embryonic stem cell (hESC) culture is routinely performed using inactivated mouse embryonic fi broblasts (MEFs) as a feeder cell layer (FL). Although these cells maintain pluripotency of hESCs, the molecular basis for this is unknown. Objectives of this study were to determine whether timing between MEF inactivation and their use as a FL infl uenced hESC growth and differentiation, and to begin defi ning the mechanism(s) involved. hESCs were plated on MEFs prepared 1 (MEF-1), 4 (MEF-4), and 7 (MEF-7) days earlier. hESC colony morphology and Oct3/4 expression levels were evaluated to determine the infl uence of different FLs. Signifi cant enhancement of hESC growth (self-renewal) was observed on MEF-1 compared with MEF-4 and/or MEF-7. Conditioned media (CM) collected from MEF-1 supported signifi cantly better hESC growth in a FL-free system compared to MEF-7 CM. Effects of MEFs on hESC growth were not caused by differences in cell density or viability, although indications of apoptosis were observed in MEF-7. Scanning electron microscopy demonstrated that MEF-7 were morphologically distinct from MEF-1 and MEF-4. Microarray analysis identifi ed 19 genes related to apoptosis with signifi cantly different levels of expression between MEF-1 and MEF-7. Several differentially expressed RNAs had gene ontology classifi cations associated with extracellular matrix (ECM) structural constituents and growth factors. Because members of Wnt signaling pathway were identifi ed in the array analysis, we examined the ability of the Wnt1 CM and secreted frizzled-related proteins to affect hESC growth and differentiation. The addition of Wnt1 CM to both MEF-1 and MEF-7 signifi cantly increased the number of undifferentiated colonies, while the addition of Sfrps promoted differentiation. Together, these results suggest that microenvironment, ECM, and soluble factors expressed by MEF-1 are signifi cantly better at maintaining self-renewal and pluripotency of hESCs. Our fi ndings have important implications in the optimization of hESC culture when MEFs are used as FL or CM is used in FL-free culture.
Human embryonic stem cells (hESCs) provided a cell source for biotechnological and clinical appli... more Human embryonic stem cells (hESCs) provided a cell source for biotechnological and clinical applications. Yet, there is limited understanding of how mechanical signals in the microenvironment of hESCs regulate their fate decisions. Here, we applied a microfabricated micromechanical platform to investigate mechanoresponsive behaviors of hESCs. We demonstrated that hESCs are mechanosensitive, and they could increase their cytoskeleton contractility with matrix rigidity. Furthermore, rigid substrates supported maintenance of pluripotency of hESCs. Matrix mechanics-mediated cytoskeleton contractility of hESCs might be functionally correlated with E-cadherin expressions in cell-cell contacts and thus involved in fate decisions of hESCs. Our results provided a novel approach to characterize and understand mechanotransduction in hESC function regulation.
Journal of The American College of Surgeons, Sep 1, 2013
A journal for the publication of original work, review and comment in the field of reproductive b... more A journal for the publication of original work, review and comment in the field of reproductive biology, reproductive endocrinology and developmental biology, including puberty, lactation and fetal physiology when they fall within these fields w w w. p u b l i s h. c s i r o. a u / j o u r n a l s / r f d All enquiries and manuscripts should be directed to Reproduction, Fertility and Development CSIRO PUBLISHING
Stem Cells and Development, Nov 1, 2014
The establishment of a reliable prenatal source of autologous, transgene-free progenitor cells ha... more The establishment of a reliable prenatal source of autologous, transgene-free progenitor cells has enormous potential in the development of regenerative-medicine-based therapies for infants born with devastating birth defects. Here, we show that a largely CD117-negative population of human amniotic fluid mesenchymal stromal cells (AF-MSCs) obtained from fetuses with or without prenatally diagnosed anomalies are readily abundant and have limited baseline differentiation potential when compared with bone-marrow-derived MSCs and other somatic cell types. Nonetheless, the AF-MSCs could be easily reprogrammed into induced pluripotent stem cells (iPSCs) using nonintegrating Sendai viral vectors encoding for OCT4, SOX2, KLF4, and cMYC. The iPSCs were virtually indistinguishable from human embryonic stem cells in multiple assays and could be used to generate a relatively homogeneous population of neural progenitors, expressing PAX6, SOX2, SOX3, Musashi-1, and PSA-NCAM, for potential use in neurologic diseases. Further, these neural progenitors showed engraftment potential in vivo and were capable of differentiating into mature neurons and astrocytes in vitro. This study demonstrates the usefulness of AF-MSCs as an excellent source for the generation of human transgene-free iPSCs ideally suited for autologous perinatal regenerative medicine applications.
Stem Cells, Dec 19, 2012
Current practices to maintain human pluripotent stem cells (hPSCs), which include induced pluripo... more Current practices to maintain human pluripotent stem cells (hPSCs), which include induced pluripotent stem cells and embryonic stem cells, in an undifferentiated state typically depend on the support of feeder cells such as mouse embryonic fibroblasts (MEFs) or an extracellular matrix such as Matrigel. Culture conditions that depend on these undefined support systems limit our ability to interpret mechanistic studies aimed at resolving how hPSCs interact with their extracellular environment to remain in a unique undifferentiated state and to make fate-changing lineage decisions. Likewise, the xenogeneic components of MEFs and Matrigel ultimately hinder our ability to use pluripo-tent stem cells to treat debilitating human diseases. Many of these obstacles have been overcome by the development of synthetic coatings and bioreactors that support hPSC expansion and self-renewal within defined culture conditions that are free from xenogeneic contamination. The establishment of defined culture conditions and synthetic matrices will facilitate studies to more precisely probe the molecular basis of pluripotent stem cell self-renewal and differentiation. When combined with three-dimensional cultures in bioreactors, these systems will also enable large-scale expansion for future clinical applications. STEM CELLS 2013;31:1-7 Disclosure of potential conflicts of interest is found at the end of this article.
Stem Cells Translational Medicine, Mar 22, 2019
Induced pluripotent stem cells (iPSCs) are cells genetically reprogrammed from somatic cells, whi... more Induced pluripotent stem cells (iPSCs) are cells genetically reprogrammed from somatic cells, which can be differentiated into neurological lineages with the aim to replace or assist damaged neurons in the treatment of spinal cord injuries (SCIs) caused by physical trauma. Here, we review studies addressing the functional use of iPSC-derived neural cells in SCIs and perform a meta-analysis to determine if significant motor improvement is restored after treatment with iPSC-derived neural cells compared with treatments using embryonic stem cell (ESC)-derived counterpart cells and control treatments. Overall, based on locomotion scales in rodents and monkeys, our meta-analysis indicates a therapeutic benefit for SCI treatment using neural cells derived from either iPSCs or ESCs, being this of importance due to existing ethical and immunological complications using ESCs. Results from these studies are evidence of the successes and limitations of iPSC-derived neural cells in the recovery of motor capacity. STEM CELLS TRANSLA-TIONAL MEDICINE 2019;00:1-13 SIGNIFICANCE STATEMENT The present review and meta-analysis evaluates the efficacy of using induced pluripotent stem cells (iPSCs)-derived neural cells in restoring motor functionality in experimental animal models sustaining traumatic spinal cord injuries. The study also addresses existing concerns with the use of iPSC-derived neural cells and whether this provides similar results as treatment with cells derived from embryonic stem cells, which have already been successfully used to treat injuries to the central nervous system.
Nanoscale, 2018
Regulation of human pluripotent stem cell (hPSC) behaviors has been mainly studied through explor... more Regulation of human pluripotent stem cell (hPSC) behaviors has been mainly studied through exploration of biochemical factors. However, current directed differentiation protocols for hPSCs that completely rely on biochemical factors remain suboptimal. It has recently become evident that coexisting biophysical signals in the stem cell microenvironment, including nanotopographic cues, can provide potent regulatory signals to mediate adult stem cell behaviors, including self-renewal and differentiation. Herein, we utilized a recently developed, large-scale nanofabrication technique based on reactive-ion etching (RIE) to generate random nanoscale structures on glass surfaces with high precision and reproducibility. We report here that hPSCs are sensitive to nanotopographic cues and such nanotopographic sensitivity can be leveraged for improving directed neuronal differentiation of hPSCs. We demonstrate early neuroepithelial conversion and motor neuron (MN) progenitor differentiation of hPSCs can be promoted using nanoengineered topographic substrates. We further explore how hPSCs sense substrate nanotopography and relay this biophysical signal through a regulatory signaling network involving cell adhesion, actomyosin cytoskeleton, and Hippo/YAP signaling to mediate neuroepithelial induction of hPSCs. Our study provides an efficient method for large-scale production of MNs from hPSCs, useful for regenerative medicine and cell-based therapies.
Biology of Reproduction, Aug 1, 2004
The p38 MAPK is a member of the mitogen-activated protein kinase (MAPK) family that participates ... more The p38 MAPK is a member of the mitogen-activated protein kinase (MAPK) family that participates in a signaling cascade in response to cytokines and stress in somatic cells. The present study was designed to investigate the expression and possible function of p38 MAPK in porcine oocytes during maturation. In immunoblots, p38 MAPK was detected in oocytes and cumulus cells. Its activity was determined during oocyte maturation in vitro by the phosphorylation of its substrate, activated transcription factor 2. As ERK1/2, oocyte p38 MAPK became active around germinal vesicle breakdown (GVBD) and maintained activity until metaphase II (MII). Immunofluorescent microscopy showed phosphorylated p38 MAPK accumulated in the nucleus before GVBD and localized in the cytoplasm and around chromosomes from metaphase I (MI) to MII. In cultured cumulusoocyte complexes, a specific inhibitor of p38 MAPK, SB203580, inhibited phosphorylation of p38 MAPK in cumulus cells and blocked both FSH-induced cumulus expansion and meiotic resumption of oocytes. During spontaneous meiotic resumption of denuded oocytes, SB203580 did not affect GVBD, but it significantly decreased the number of oocytes reaching MII and conversely increased the number of oocytes arrested at MI. These results suggest that p38 MAPK in porcine oocytes becomes active around GVBD, remains active through MI to MII, and has a role in MI-MII transition, and that cumulus p38 MAPK might be involved in FSH-induced meiotic resumption of oocytes.
Reproduction, Fertility and Development, 2004
The importance of protein phosphorylations during meiotic maturation (transition from prophase I ... more The importance of protein phosphorylations during meiotic maturation (transition from prophase I to metaphase II) of oocytes is documented by the fact that the inhibition of the M-phase kinases, cdc2k or MAPK, arrests the oocytes in the GV stage. A detailed knowledge of the targets of these kinases during this stage of development is still missing. Therefore, we have analyzed the proteome of bovine oocytes by high resolution 2D-gel electrophoresis to detect differences in the expression and phosphorylation state of proteins in the course of in vitro maturation (IVM). Bovine oocytes were matured for different times in TCM 199 containing 3% BSA and 300 oocytes each in GV stage (0-h maturation), in GVBD/M I (10-h maturation) or in M II stage (240 h maturation) were separated on the gels. The proteins were visualized by staining them with silver or with the fluorescence dye Sypro Ruby, and phosphorylated proteins were detected by Western Blotting with Ser-, Thr-, or Tyr-phosphorylation specific antibodies or by staining with the phosphoprotein specific fluorescence dye Pro-Q Diamond. Gels made from oocytes at the above mentioned maturation stages were compared by a computerized gel-overlay software program (2D Decodon, Greitswald, Germany). The overall protein synthesis was statistically analysed by ANOVA (SigmaStat, Ekrath, Germany), pairwise multiple comparison procedure. Only distinct spots with a difference greater than 30% in their optical densities were considered to be differently expressed or phosphorylated. The results showed a threefold increase in the rate of overall protein synthesis (p 0.05) during GVBD. Newly synthesized proteins were detected mainly in the higher molecular weight (MW) range (60-80 kDa), and protein degradations were found mainly in the lower MW range (20-40 kDa) after GVBD. Preliminary data obtained by analyzing the phosphorylation pattern showed that obviously no phosphorylated proteins could be detected in the GV-stage oocytes. Phosphorylation of different proteins was observed at the time of GVBD after 6 to 10 h IVM, concomitantly with the activation of cdc2k and MAPK. A maximum of phosphorylated proteins was observed in metaphase II. The first results obtained by performing peptide mass fingerprinting using MALDI-Tof showed that members of the family of heat-shock proteins, ribosomal proteins and putative zinc finger proteins (transcription regulators) were differently expressed or phosphorylated during IVM.
ASME 2012 Summer Bioengineering Conference, Parts A and B, Jun 20, 2012
ABSTRACT Much effort has been recently directed to investigate how soluble factors in the local c... more ABSTRACT Much effort has been recently directed to investigate how soluble factors in the local cellular microenvironment of embryonic stem cells (ESCs) regulate their fate decisions [1]; however, effects of mechanical signals in the local cellular microenvironment on the fates of ESCs are still not yet well understood. Early experimental evidence established in recent years has shown that mechanical signals and external forces experienced through interactions with extracellular matrix (ECM) mechanics can play critical roles in regulating survival, proliferation and differentiation of ESCs. However, there is still limited knowledge of how mechanical signals in the local cellular microenvironment regulate the fate decisions of human ESCs (hESCs), and advancing such knowledge will be critical for both fundamental understanding and clinical applications of hESCs. This work was thus set to explicitly investigate the mechanosensitive properties of hESCs.
Biology of Reproduction, Jul 1, 2007
Bioengineering
Human embryonic stem cells and induced pluripotent stem cells (hPSC) have an unprecedented opport... more Human embryonic stem cells and induced pluripotent stem cells (hPSC) have an unprecedented opportunity to revolutionize the fields of developmental biology as well as tissue engineering and regenerative medicine. However, their applications have been significantly limited by the lack of chemically defined and xeno-free culture conditions. The demand for the high-quality and scaled-up production of cells for use in both research and clinical studies underscores the need to develop tools that will simplify the in vitro culture process while reducing the variables. Here, we describe a systematic study to identify the optimal conditions for the initial cell attachment of hPSC to tissue culture dishes grafted with polymers of N-(3-Sulfopropyl)-N-Methacryloxyethyl-N, N-Dimethylammoniun Betaine (PMEDSAH) in combination with chemically defined and xeno-free culture media. After testing multiple supplements and chemicals, we identified that pre-conditioning of PMEDSAH grafted plates with 10%...
Cancers
Over the past decades, our knowledge of integrins has evolved from being understood as simple cel... more Over the past decades, our knowledge of integrins has evolved from being understood as simple cell surface adhesion molecules to receptors that have a complex range of intracellular and extracellular functions, such as delivering chemical and mechanical signals to cells. Consequently, they actively control cellular proliferation, differentiation, and apoptosis. Dysregulation of integrin signaling is a major factor in the development and progression of many tumors. Many reviews have covered the broader integrin family in molecular and cellular studies and its roles in diseases. Nevertheless, further understanding of the mechanisms specific to an individual subunit of different heterodimers is more useful. Thus, we describe the current understanding of and exploratory investigations on the α6-integrin subunit (CD49f, VLA6; encoded by the gene itga6) in normal and cancer cells. The roles of ITGA6 in cell adhesion, stemness, metastasis, angiogenesis, and drug resistance, and as a diagno...
Journal of Stem Cell Research & Therapy, Nov 7, 2013
npj Microgravity
A systematic study on the biological effects of simulated microgravity (sµg) on human pluripotent... more A systematic study on the biological effects of simulated microgravity (sµg) on human pluripotent stem cells (hPSC) is still lacking. Here, we used a fast-rotating 2-D clinostat to investigate the sµg effect on proliferation, self-renewal, and cell cycle regulation of hPSCs. We observed significant upregulation of protein translation of pluripotent transcription factors in hPSC cultured in sµg compared to cells cultured in 1g conditions. In addition to a significant increase in expression of telomere elongation genes. Differentiation experiments showed that hPSC cultured in sµg condition were less susceptible to differentiation compared to cells in 1g conditions. These results suggest that sµg enhances hPSC self-renewal. Our study revealed that sµg enhanced the cell proliferation of hPSCs by regulating the expression of cell cycle-associated kinases. RNA-seq analysis indicated that in sµg condition the expression of differentiation and development pathways are downregulated, while m...
The Journal of craniofacial surgery (Print), 2012
Gene therapy in the craniofacial region provides a unique tool for delivery of DNA to coordinate ... more Gene therapy in the craniofacial region provides a unique tool for delivery of DNA to coordinate protein production in both time and space. The drive to bring this technology to the clinic is derived from the fact that over 85% of the global population may at one time require repair or replacement of a craniofacial structure. This need ranges from mild tooth decay and tooth loss to temporomandibular joint disorders and large-scale reconstructive surgery. Our ability to insert foreign DNA into a host cell has been developing since early uses of gene therapy to alter bacterial properties for waste cleanup in the 1980s followed by successful human clinical trials in the 1990s to treat severe combined immunodeficiency. In the past twenty years the emerging field of craniofacial tissue engineering has adopted these techniques to enhance regeneration of mineralized tissues, salivary gland, periodontium, and to reduce tumor burden of head and neck squamous cell carcinoma. Studies are currently pursuing research on both biomaterial-mediated gene delivery as well as more clinically efficacious, though potentially more hazardous, viral methods. Though hundreds of gene therapy clinical trials have taken place in the past twenty years, we must still work to ensure an ideal safety profile for each gene and delivery method combination. With adequate genotoxicity testing, we can expect gene therapy to augment protein delivery strategies and potentially allow for tissue-specific targeting, delivery of multiple signals, and increased spatial and temporal control with the goal of natural tissue replacement in the craniofacial complex.
British Journal of Cancer, 2021
BACKGROUND Bone-marrow-derived haematopoietic stem and progenitor cells (HSPCs) are a prominent p... more BACKGROUND Bone-marrow-derived haematopoietic stem and progenitor cells (HSPCs) are a prominent part of the highly complex tumour microenvironment (TME) where they localise within tumours and maintain haematopoietic potency. Understanding the role HSPCs play in tumour growth and response to radiation therapy (RT) may lead to improved patient treatments and outcomes. METHODS We used a mouse model of non-small cell lung carcinoma where tumours were exposed to RT regimens alone or in combination with GW2580, a pharmacological inhibitor of colony stimulating factor (CSF)-1 receptor. RT-PCR, western blotting and immunohistochemistry were used to quantify expression levels of factors that affect HSPC differentiation. DsRed+ HSPC intratumoural activity was tracked using flow cytometry and confocal microscopy. RESULTS We demonstrated that CSF-1 is enhanced in the TME following exposure to RT. CSF-1 signaling induced intratumoural HSPC differentiation into M2 polarised tumour-associated macrophages (TAMs), aiding in post-RT tumour survival and regrowth. In contrast, hyperfractionated/pulsed radiation therapy (PRT) and GW2580 ablated this process resulting in improved tumour killing and mouse survival. CONCLUSIONS Tumours coopt intratumoural HSPC fate determination via CSF-1 signaling to overcome the effects of RT. Thus, limiting intratumoural HSPC activity represents an attractive strategy for improving the clinical treatment of solid tumours.
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Papers by Luis Villa-Diaz