Papers by William Stanford
PLOS ONE, Aug 4, 2009
Background: Mesenchymal progenitor cells (MPCs) have been isolated from a variety of connective t... more Background: Mesenchymal progenitor cells (MPCs) have been isolated from a variety of connective tissues, and are commonly called ''mesenchymal stem cells'' (MSCs). A stem cell is defined as having robust clonal self-renewal and multilineage differentiation potential. Accordingly, the term ''MSC'' has been criticised, as there is little data demonstrating self-renewal of definitive single-cell-derived (SCD) clonal populations from a mesenchymal cell source. Methodology/Principal Findings: Here we show that a tractable MPC population, human umbilical cord perivascular cells (HUCPVCs), was capable of multilineage differentiation in vitro and, more importantly, contributed to rapid connective tissue healing in vivo by producing bone, cartilage and fibrous stroma. Furthermore, HUCPVCs exhibit a high clonogenic frequency, allowing us to isolate definitive SCD parent and daughter clones from mixed gender suspensions as determined by Y-chromosome fluorescent in situ hybridization. Conclusions/Significance: Analysis of the multilineage differentiation capacity of SCD parent clones and daughter clones enabled us to formulate a new hierarchical schema for MSC self-renewal and differentiation in which a self-renewing multipotent MSC gives rise to more restricted self-renewing progenitors that gradually lose differentiation potential until a state of complete restriction to the fibroblast is reached.
Genes & Development, Jan 22, 2003
Mouse embryos mutant for the VEGF receptor, VEGFR2, Flk-1, or Kdr, fail to form both endothelial ... more Mouse embryos mutant for the VEGF receptor, VEGFR2, Flk-1, or Kdr, fail to form both endothelial and hematopoietic cells, suggesting a possible role in a common progenitor to both lineages. The transcription factor Tal1 (Scl), is not expressed in Flk1 −/− embryos, consistent with a downstream role in the Flk1 pathway. We tested whether expression of Tal1 under the Flk1 promoter was sufficient to rescue the loss of endothelial and hematopoietic cells in Flk1 mutants. Only partial rescue of hematopoiesis and endothelial development was observed in vivo. However, Flk1 −/Tal1 embryonic stem (ES) cells were capable of blast colony formation in vitro at levels equivalent to Flk1 +/− heterozygotes. Ectopic expression of Tal1 under the Flk1 promoter in Flk1 +/− mouse embryos or ES cells caused no obvious pathology but increased the number of blast colony forming cells (BL-CFCs) and enhanced their hematopoietic potential. These single-cell-derived BL-CFCs also produced smooth muscle cells in vitro. Increased Tal1 expression inhibited smooth muscle differentiation in this assay, whereas loss of Tal1 promoted smooth muscle formation. We propose a model in which the combinatorial effects of Flk1 and Tal1 act to regulate cell fate choice in early development into hematopoietic, endothelial, and smooth muscle lineages.
Cell discovery, Jan 22, 2019
Metacaspase enzymes are critical regulatory factors that paradoxically engage apoptosis and also ... more Metacaspase enzymes are critical regulatory factors that paradoxically engage apoptosis and also maintain cell viability. For example, the Saccharomyces cerevisiae metacaspase Yca1 has been shown to be important for maintaining cellular proteostasis during stress, and the loss of this enzyme results in increased retention of aggregated material within the insoluble proteome. However, the molecular mechanism(s) by which Yca1 maintains cellular proteostasis remains unknown. Here, using proteomic analysis coupled with protein interaction studies we identified a direct interplay between Yca1 and the ubiquitin-proteasome system. We noted multiple ubiquitination sites on Yca1 and established Rsp5 as the candidate E3 ligase involved in this process. Further characterization of the ubiquitination sites identified the K355 residue on Yca1 as a critical modification for proteostasis function, managing both insoluble protein content and vacuolar response. We also identified a Yca1 phosphorylation site at S346, which promoted interaction with Rsp5 and the aggregate dispersal function of the metacaspase. Interestingly, proteomic analysis also revealed that Yca1 interacts with the ubiquitin precursor protein Rps31, cleaving the protein to release free ubiquitin. In turn, loss of Yca1 or its catalytic activity reduced the levels of monomeric ubiquitin in vivo, concurrent to increased protein aggregation. The K355 and S346 residues were also observed to influence the abundance of low-molecular weight ubiquitin. Together, these observations suggest that Yca1 maintains proteostasis and limits protein aggregation by ensuring a free flow of monoubiquitin, an essential precursor for ligase-enhanced Yca1 enzymatic activity and general proteasome-mediated protein degradation.
Biology of Reproduction, Oct 1, 2005
Recently, it was found by two research groups that LY6A, known widely in the stem cell community ... more Recently, it was found by two research groups that LY6A, known widely in the stem cell community as stem cell antigen-1 or SCA-1, is expressed on testicular side population (SP) cells. Whether these SP cells are spermatogonial stem cells is a point of disagreement and, therefore, the identity of the LY6A-positive cells as well. We studied the expression pattern of LY6A in testis by immunohistochemistry and found it to be expressed in the interstitial tissue on peritubular myoid, endothelial, and spherical-shaped peritubular mesenchymal cells. To address the question whether LY6A has a function in spermatogenesis or testis development, we studied the testis of Ly6a ؊/؊ mice (allele Ly6a tm1Pmf). We found no morphological abnormalities or differences in numbers of spermatogonia, spermatocytes, Leydig cells, or macrophages in relation to the number of Sertoli cells. Therefore, we conclude that LY6A expression does not influence testis development or spermatogenesis and that spermatogonial stem cells are LY6A negative.
Circulation, Mar 23, 2004
Cell discovery, May 1, 2018
Polycomb repressive complex 2 (PRC2) accessory proteins play substoichiometric, tissue-specific r... more Polycomb repressive complex 2 (PRC2) accessory proteins play substoichiometric, tissue-specific roles to recruit PRC2 to specific genomic loci or increase enzymatic activity, while PRC2 core proteins are required for complex stability and global levels of trimethylation of histone 3 at lysine 27 (H3K27me3). Here, we demonstrate a role for the classical PRC2 accessory protein Mtf2/Pcl2 in the hematopoietic system that is more akin to that of a core PRC2 protein. Mtf2 −/− erythroid progenitors demonstrate markedly decreased core PRC2 protein levels and a global loss of H3K27me3 at promoter-proximal regions. The resulting de-repression of transcriptional and signaling networks blocks definitive erythroid development, culminating in Mtf2 −/− embryos dying by e15.5 due to severe anemia. Gene regulatory network (GRN) analysis demonstrated Mtf2 directly regulates Wnt signaling in erythroblasts, leading to activated canonical Wnt signaling in Mtf2-deficient erythroblasts, while chemical inhibition of canonical Wnt signaling rescued Mtf2-deficient erythroblast differentiation in vitro. Using a combination of in vitro, in vivo and systems analyses, we demonstrate that Mtf2 is a critical epigenetic regulator of Wnt signaling during erythropoiesis and recast the role of polycomb accessory proteins in a tissue-specific context.
Cancer Discovery, Nov 1, 2018
Deep sequencing has revealed that epigenetic modifi ers are the most mutated genes in acute myelo... more Deep sequencing has revealed that epigenetic modifi ers are the most mutated genes in acute myeloid leukemia (AML). Thus, elucidating epigenetic dysregulation in AML is crucial to understand disease mechanisms. Here, we demonstrate that metal response element binding transcription factor 2/polycomblike 2 (MTF2/PCL2) plays a fundamental role in the polycomb repressive complex 2 (PRC2) and that its loss elicits an altered epigenetic state underlying refractory AML. Unbiased systems analyses identifi ed the loss of MTF2-PRC2 repression of MDM2 as central to, and therefore a biomarker for, refractory AML. Thus, immature MTF2-defi cient CD34 + CD38 − cells overexpress MDM2, thereby inhibiting p53 that leads to chemoresistance due to defects in cellcycle regulation and apoptosis. Targeting this dysregulated signaling pathway by MTF2 overexpression or MDM2 inhibitors sensitized refractory patient leukemic cells to induction chemotherapeutics and prevented relapse in AML patient-derived xenograft mice. Therefore, we have uncovered a direct epigenetic mechanism by which MTF2 functions as a tumor suppressor required for AML chemotherapeutic sensitivity and identifi ed a potential therapeutic strategy to treat refractory AML. SIGNIFICANCE: MTF2 defi ciency predicts refractory AML at diagnosis. MTF2 represses MDM2 in hematopoietic cells and its loss in AML results in chemoresistance. Inhibiting p53 degradation by overexpressing MTF2 in vitro or by using MDM2 inhibitors in vivo sensitizes MTF2-defi cient refractory AML cells to a standard induction-chemotherapy regimen. Cancer Discov; 8(11); 1376-89. ©2018 AACR.
PLOS ONE, Sep 27, 2019
Human embryonic stem cell (hESC)-derived skeletal muscle progenitors (SMP)-defined as PAX7-expres... more Human embryonic stem cell (hESC)-derived skeletal muscle progenitors (SMP)-defined as PAX7-expressing cells with myogenic potential-can provide an abundant source of donor material for muscle stem cell therapy. As in vitro myogenesis is decoupled from in vivo timing and 3D-embryo structure, it is important to characterize what stage or type of muscle is modeled in culture. Here, gene expression profiling is analyzed in hESCs over a 50 day skeletal myogenesis protocol and compared to datasets of other hESC-derived skeletal muscle and adult murine satellite cells. Furthermore, day 2 cultures differentiated with high or lower concentrations of CHIR99021, a GSK3A/GSK3B inhibitor, were contrasted. Expression profiling of the 50 day time course identified successively expressed gene subsets involved in mesoderm/paraxial mesoderm induction, somitogenesis, and skeletal muscle commitment/formation which could be regulated by a putative cascade of transcription factors. Initiating differentiation with higher CHIR99021 concentrations significantly increased expression of MSGN1 and TGFB-superfamily genes, notably NODAL, resulting in enhanced paraxial mesoderm and reduced ectoderm/neuronal gene expression. Comparison to adult satellite cells revealed that genes expressed in 50-day cultures correlated better with those expressed by quiescent or early activated satellite cells, which have the greatest therapeutic potential. Day 50 cultures were similar to other hESC-derived skeletal muscle and both expressed known and novel SMP surface proteins. Overall, a putative cascade of transcription factors has been identified which regulates four stages of myogenesis. Subsets of these factors were upregulated by high CHIR99021 or their binding sites were
Scientific Reports, Apr 22, 2020
Trophoblast-like DNA methylation profile of macTSCs. Recently, Lee et al. proposed a criteria tha... more Trophoblast-like DNA methylation profile of macTSCs. Recently, Lee et al. proposed a criteria that involves hypomethylation of the ELF5 promoter to define human early trophoblast cells 22. We also identified differentially methylated genomic regions, with higher methylation in the trophoblast cell lineage than in the embryonic cell lineage in mice and humans, and named such regions trophoblast-embryonic tissue-dependent and differentially methylated regions (T-E T-DMRs) 23. To characterize macTSCs, we analyzed the DNA methylation status of the ELF5 promoter, and the T-E T-DMRs by bisulfite sequencing (Fig. 2). The OCT4 promoter region was hypermethylated, while the ELF5 promoter was hypomethylated in macTSC#2 (Fig. 2A), demonstrating that macTSC possess trophoblastic DNA methylation status.
Parkinsona’s disease (PD) is a debilitating neurodegenerative disease characterized by the loss o... more Parkinsona’s disease (PD) is a debilitating neurodegenerative disease characterized by the loss of midbrain dopaminergic neurons (DaNs) and the abnormal accumulation of α-Synuclein (α-Syn) protein. Currently, no treatment can slow nor halt the progression of PD. Multiplications and mutations of the α-Syn gene (SNCA) cause PD-associated syndromes and animal models that overexpress α-Syn replicate several features of PD. Decreasing total α-Syn levels, therefore, is an attractive approach to slow down neurodegeneration in patients with synucleinopathy. We previously performed a genetic screen for modifiers of α-Syn levels and identified CDK14, a kinase of largely unknown function as a regulator of α-Syn. To test the potential therapeutic effects of CDK14 reduction in PD, we ablated Cdk14 in the α-Syn preformed fibrils (PFF)-induced PD mouse model. We found that loss of Cdk14 mitigates the grip strength deficit of PFF-treated mice and ameliorates PFF-induced cortical α-Syn pathology, in...
Methods in Molecular Biology, 2009
Background: A number of cardiovascular, neurological, musculoskeletal and other diseases have a l... more Background: A number of cardiovascular, neurological, musculoskeletal and other diseases have a limited capacity for repair and only a modest progress has been made in treatment of brain diseases. The discovery of stem cells has opened new possibilities for the treatment of these maladies, and cell therapy now stands at the cutting-edge of modern regenerative medicine and tissue engineering. Experimental data and the first clinical trials employing stem cells have shown their broad therapeutic potential and have brought hope to patients suffering from devastating pathologies of different organs and systems. Aims: Here, we briefly review the main achievements and trends in cell-based therapy, with an emphasis on the main types of stem cells: embryonic, mesenchymal stromal and induced pluripotent cells. Discussion: Many questions regarding the application of stem cells remain unanswered, particularly tumorigenicity, immune rejection and danger of gene manipulation. Currently, only MSC seems to be safe and might be considered to be a leading candidate for human application to treat pathologies that affect the cardiovascular, neurological and musculoskeletal systems.
Circulation Research, Sep 15, 2006
Evidence suggests that bone marrow (BM) cells may give rise to a significant proportion of smooth... more Evidence suggests that bone marrow (BM) cells may give rise to a significant proportion of smooth muscle cells (SMCs) that contribute to intimal hyperplasia after vascular injury; however, the molecular pathways involved and the timeline of these events remain poorly characterized. We hypothesized that the stem cell factor (SCF)/c-Kit tyrosine kinase signaling pathway is critical to neointimal formation by BM-derived progenitors. Wire-induced femoral artery injury in mice reconstituted with wild-type BM cells expressing yellow fluorescent protein was performed, which revealed that 66Ϯ12% of the SMCs (␣-smooth muscle actin-positive [␣SMA ϩ ] cells) in the neointima were from BM. To characterize the role of the SCF/c-Kit pathway, we used c-Kit deficient W/W v and SCF-deficient Steel-Dickie mice. Strikingly, vascular injury in these mice resulted in almost a complete inhibition of neointimal formation, whereas wild-type BM reconstitution of c-Kit mutant mice led to neointimal formation in a similar fashion as wild-type animals, as did chronic administration of SCF in matrix metalloproteinase-9-deficient mice, a model of soluble SCF deficiency. Pharmacological antagonism of the SCF/c-Kit pathway with imatinib mesylate (Gleevec) or ACK2 (c-Kit antibody) also resulted in a marked reduction in intimal hyperplasia. Vascular injury resulted in the local upregulation of SCF expression. c-Kit ϩ progenitor cells (PCs) homed to the injured vascular wall and differentiated into ␣SMA ϩ cells. Vascular injury also caused an increase in circulating SCF levels which promoted CD34 ϩ PC mobilization, a response that was blunted in mutant and imatinib mesylate-treated mice. In vitro, SCF promoted adhesion of BM PCs to fibronectin. Additionally, anti-SCF antibodies inhibited adhesion of BM PCs to activated SMCs and diminished SMC differentiation. These data indicate that SCF/c-Kit signaling plays a pivotal role in the development of neointima by BM-derived PCs and that the inhibition of this pathway may serve as a novel therapeutic target to limit aberrant vascular remodeling.
Human pluripotent stem cells (hPSCs) are an essential cell source in tissue engineering, studies ... more Human pluripotent stem cells (hPSCs) are an essential cell source in tissue engineering, studies of development, and disease modeling. Efficient, broadly amenable protocols for rapid lineage induction of hPSCs are of great interest in the stem cell biology field. We describe a simple, robust method for differentiation of hPSCs into mesendoderm in defined conditions utilizing single-cell seeding (SCS) and BMP4 and Activin A (BA) treatment. Gene sets and gene ontology terms related to mesoderm and endoderm differentiation were enriched after 48 hours of BA treatment. BA treatment was readily incorporated into existing protocols for chondrogenic and endothelial progenitor cell differentiation. After prolonged differentiation in vitro or in vivo, BA pre-treatment resulted in higher mesoderm and endoderm levels at the expense of ectoderm formation. These data demonstrate that SCS with BA treatment is a powerful method for induction of mesendoderm that can be integrated into protocols for mesoderm and endoderm differentiation.
Nature Genetics, Jun 1, 2004
Gene trapping is a high-throughput approach that can be used to introduce insertional mutations a... more Gene trapping is a high-throughput approach that can be used to introduce insertional mutations across the genome in mouse embryonic stem (ES) cells. Gene trap vectors simultaneously mutate and report the expression of the endogenous gene at the site of insertion and provide a DNA tag for the rapid identification of the disrupted gene. The generation of mutant mice from a large collection of ES cell lines carrying gene trap insertions could be applied to large-scale functional analysis of the ∼30,000 mammalian genes. The overall impact of gene trap resources will rest on the fraction of the genome that is accessible with this technology, the efficiency relative to other competing technologies and the availability of such a resource to the academic community. Lexicon Genetics, a US-based biotechnology company, was the first to implement a genome-wide gene trapping program 1 and has developed OmniBank (http://www.lexicongenetics.com), the largest library of mutant ES cell lines. A parallel effort was initiated in the public domain by several academic groups in the International Gene Trap Consortium (IGTC; http://www.igtc.ca). The recent release of OmniBank sequence tags to GenBank 2 has made it possible to compare the size and efficiency of the existing gene trap libraries. We confirm that Lexicon achieved close to 60% coverage of the genome from 200,000 OmniBank sequence tags deposited in GenBank (Fig. 1). Our analysis, supported independently by Lexicon 3 , indicates that the rate of trapping new genes was not linear but declined within the first 100,000 tags to a rate at which 1 new gene was added every 35 tags, comparable to the efficiency of high-throughput gene targeting methods 4. To date, the IGTC has attained 32% genome coverage in 27,000 tags; trapping
Science, Feb 16, 2001
With the reports of the DNA sequence of the human genome and progress in sequencing the mouse gen... more With the reports of the DNA sequence of the human genome and progress in sequencing the mouse genome, the first phase of the Human Genome Project is complete (13)). Analysis of these DNA sequences will reveal the inventory of genes used for building these ...
Stem Cells, Mar 22, 2007
Cloned 20 years ago, stem cell antigen-1 (Sca-1) is used extensively to enrich for murine hematop... more Cloned 20 years ago, stem cell antigen-1 (Sca-1) is used extensively to enrich for murine hematopoietic stem cells. The realization that many different stem cell types share conserved biochemical pathways has led to a flood of recent research using Sca-1 as a candidate marker in the search for tissue-resident and cancer stem cells. Although surprisingly little is still known about its biochemical function, the generation and analysis of knockout mice has begun to shed light on the functions of Sca-1 in stem and progenitor cells, demonstrating that it is more than a convenient marker for stem cell biologists. This review summarizes the plethora of recent findings utilizing Sca-1 as a parenchymal stem cell marker and detailing its functional role in stem and progenitor cells and also attempts to explain the lingering mysteries surrounding its biochemical function and human ortholog.
Stem cell reports, Dec 1, 2019
Human pluripotent stem cells (hPSCs) are an essential cell source in tissue engineering, studies ... more Human pluripotent stem cells (hPSCs) are an essential cell source in tissue engineering, studies of development, and disease modeling. Efficient, broadly amenable protocols for rapid lineage induction of hPSCs are of great interest in the stem cell biology field. We describe a simple, robust method for differentiation of hPSCs into mesendoderm in defined conditions utilizing single-cell seeding (SCS) and BMP4 and Activin A (BA) treatment. BA treatment was readily incorporated into existing protocols for chondrogenic and endothelial progenitor cell differentiation, while fine-tuning of BA conditions facilitated definitive endoderm commitment. After prolonged differentiation in vitro or in vivo, BA pretreatment resulted in higher mesoderm and endoderm levels at the expense of ectoderm formation. These data demonstrate that SCS with BA treatment is a powerful method for induction of mesendoderm that can be adapted for use in mesoderm and endoderm differentiation.
Proceedings of the National Academy of Sciences of the United States of America, Jun 22, 1999
Genetic studies in mice have previously demonstrated an intrinsic requirement for the vascular en... more Genetic studies in mice have previously demonstrated an intrinsic requirement for the vascular endothelial growth factor (VEGF) receptor Flk-1 in the early development of both the hematopoietic and endothelial cell lineages. In this study, embryonic stem (ES) cells homozygous for a targeted null mutation in flk-1 (flk-1 (؊͞؊)) were examined for their hematopoietic potential in vitro during embryoid body (EB) formation or when cultured on the stromal cell line OP9. Surprisingly, in EB cultures flk-1 (؊͞؊) ES cells were able to differentiate into all myeloid-erythroid lineages, albeit at half the frequency of heterozygous lines. In contrast, although flk-1 (؊͞؊) ES cells formed mesodermal-like colonies on OP9 monolayers, they failed to generate hematopoietic clusters even in the presence of exogenous cytokines. However, flk-1 (؊͞؊) OP9 cultures did contain myeloid precursors, albeit at greatly reduced percentages. This defect was rescued by first allowing flk-1 (؊͞؊) ES cells to differentiate into EBs and then passaging these cells onto OP9 stroma. Thus, the requirement for Flk-1 in early hematopoietic development can be abrogated by alterations in the microenvironment. This finding is consistent with a role for Flk-1 in regulating the migration of early mesodermally derived precursors into a microenvironment that is permissive for hematopoiesis.
We identified Ras guanine-releasing protein 3 (RasGRP3) as a guanine exchange factor expressed in... more We identified Ras guanine-releasing protein 3 (RasGRP3) as a guanine exchange factor expressed in blood vessels via an embryonic stem (ES) cell-based gene trap screen to identify novel vascular genes. RasGRP3 is expressed in embryonic blood vessels, down-regulated in mature adult vessels, and reexpressed in newly formed vessels during pregnancy and tumorigenesis. This expression pattern is consistent with an angiogenic function for RasGRP3. Although a loss-of-function mutation in RasGRP3 did not affect viability, RasGRP3 was up-regulated in response to vascular endothelial growth factor (VEGF) stimulation of human umbilical vein endothelial cells, placing RasGRP3 regulation downstream of VEGF signaling. Phorbol esters mimic the second messenger diacylglycerol (DAG) in activating both protein kinase C (PKC) and non-PKC phorbol ester receptors such as RasGRP3. ES cell-derived wild-type blood vessels exposed to phorbol myristate acetate (PMA) underwent extensive aberrant morphogenesis that resulted in the formation of large endothelial sheets rather than properly branched vessels. This response to PMA was completely dependent on the presence of RasGRP3, as mutant vessels were refractory to the treatment. Taken together, these findings show that endothelial RasGRP3 is up-regulated in response to VEGF stimulation and that RasGRP3 functions as an endothelial cell phorbol ester receptor in a pathway whose stimulation perturbs normal angiogenesis. This suggests that RasGRP3 activity may exacerbate vascular complications in diseases characterized by excess DAG, such as diabetes.
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Papers by William Stanford