Megan T. Baldridge, Ph.D.
Washington University School of Medicine
Bone marrow suppression is a common adverse effect of long-term antibiotic administration, which can, in turn, leave patients at substantial risk for future infections. We previously uncovered depletion of commensal intestinal bacteria as a proximal cause of antibiotic-mediated bone marrow suppression, implicating the microbiome in maintenance of normal hematopoiesis. Commensal bacteria, acting via STAT1, a major transcription factor downstream of interferon (IFN) signaling, are necessary to promote the normal function of hematopoietic progenitors in the bone marrow. We have recently further uncovered type I IFN signaling as the critical IFN signaling pathway for this effect, and continue to gain further insight into the mechanisms by which the intestinal microbiota signals to the distal compartment of the bone marrow, with the ultimate goal of designing preventive and therapeutic approaches to combat antibiotic-associated bone marrow suppression.
Sarah Bowling, Ph.D.
Tracing the origin of cell types is key to understanding diverse, fundamental questions in biology. To date, lineage tracing research has been hindered by the technically difficult and time-consuming task of labeling and tracking individual cell populations. Here, we present a new mouse model that can be used to simultaneously interrogate the lineage and gene expression information of single cells in vivo. This mouse model, named CARLIN (CRISPR Array Repair LINeage tracing), exploits CRISPR-Cas9 technology to generate inducible, transcribed barcodes that can be detected in an unbiased and global manner. We demonstrate that CARLIN mice can be used to generate up to 30,000 unique barcodes in the mouse, and that these barcodes can be detected and read using single-cell droplet sequencing approaches. We also find that multiple pulses of labeling can be used to enhance our understanding of tissue phylogeny. Finally, we have applied our tools to investigate the clonal dynamics of hematopoiesis following chemotherapy treatment to find that recovery is driven largely by expansion of a small number of highly active HSC clones. Our work represents a new resource in lineage tracing research and sheds light on the dynamics of stem cell differentiation in the blood following perturbation.
Jingli Cai, Ph.D.
Vickie & Jack Farber Institute for Neuroscience
Thomas Jefferson University
Parkinson Disease (PD) is the second most common neurodegenerative disorder, with no cure available. The majority of midbrain dopaminergic (mDA, labeled by tyrosine hydroxylase, TH) neurons of PD patients are lost when symptoms are identified. Replacing the lost mDA neurons becomes the most promising therapeutic solution. Ever since reprogramming of adult somatic cells into induced pluripotent stem cells (iPS) became a common laboratory practice, finding the optimal protocol to differentiate iPS cells into authentic mDA neurons became increasingly important. Our laboratory has been working on developing a reliable differentiation protocol, which would give rise to Lmx1a/Foxa2/En1 positive TH neurons as those found in the mouse developing brains. Learning from the rodent embryonic brain, we found that in addition to increasing these three fate genes, eliminating other subtypes of neurons (Glutamatergic, GABAergic) in the ventral mDA area is also essential. To achieve these ends, the fine balance between SHH and Wnt signaling pathways has become especially important both to drive the correct mDA phenotype and to reduce other unwanted neurotransmitter types.
Bin Duan, Ph.D.
University of Nebraska Medical Center
Human induced pluripotent stem cells (hiPSC) not only have great potentials for therapeutic applications but also offer unprecedented promise in disease modeling and drug screening. However, currently available biomaterials that can accommodate hiPSC derived cells are inadequate and how microenvironments affect hiPSC differentiation is still inclusive. In addition, more advanced biomedical engineering techniques are needed to facilitate the application of hiPSC derived cells and models. To this end, our lab focuses on development of biomaterials and biofabrication techniques to control hiPSC fate and support the generation of hiPSC-based models. In this talk, I’ll briefly present our recent work about the effects of biomaterial-based microenvironments on hiPSC derived neural progenitor cell differentiation. Then, I’ll talk about how we use 3D printing technique to facilitate the development of hiPSC based in vitro brain-blood barrier model and moderate traumatic injury model.
Martine Dunnwald, Ph.D.
Department of Anatomy and Cell Biology,
University of Iowa
Morphogenesis of the palate requires a spatially controlled set of events that include proliferation and migration of epithelial cells. Cutaneous tissue repair requires similar cellular processes, suggesting that they may share common molecular pathways. We identified Interferon Regulatory Factor 6 (IRF6) as a transcription factor that regulates both palatogenesis and tissue repair. In particular, individuals with mutations in IRF6 (van der Woude syndrome, the most common form of syndromic orofacial cleft) exhibited increased likelihood of wound healing complications following surgical repair. We used in vitro keratinocyte cultures and in vivo genetically modified mice to further investigate the molecular signaling beyond this clinical finding. Our data demonstrate that IRF6 is required for epithelial proliferation, migration, and adhesion via the RhoA pathway. One of the downstream targets of IRF6 is Rho GTPase Activating Protein 29 (ARHGAP29). Interestingly, genetic variants in ARHGAP29 are associated with orofacial cleft. This further validates the concept that palatogenesis and wound healing share similar biological processes and are two faces of the same coin.
Supported by NIH AR067739
Novel chromatin-associated factors Dppa2/4 facilitate epigenetic remodeling during reprogramming to pluripotency
Natalia Ivanova, PhD
Department of Genetics, Yale University
Pluripotent stem cells can self-renew in culture while retaining the potential to form the full spectrum of cell lineages found in the body. Pluripotency can now be induced in fully differentiated somatic cells with four transcription factors: Oct4, Klf4, Sox2 and Myc (OKSM), yet the efficiency is low and the mechanistic understanding of the reprogramming process remains incomplete. We have recently identified chromatin-associated ES cell-specific factors Dppa2 and Dppa4 as key regulators of reprogramming process. Dppa2 and Dppa4 are induced in reprogramming intermediates, function as a heterodimer and are required for efficient reprogramming of mouse and human cells. When co-expressed with OKSM factors, Dppa2/4 yield reprogramming efficiencies that exceed 80% and accelerate reprogramming kinetics, generating iPSCs in two to four days. When bound to chromatin, Dppa2/4 initiate global chromatin decompaction via the DNA damage response pathway, contribute to down-regulation of somatic genes and activation of ESC enhancers, all of which enables an efficient transition to pluripotency. Our work provides critical insights into how the epigenome is remodeled during acquisition of pluripotency.
Cassandra Juran, Ph.D.
NASA Postdoctoral Program Fellow
NASA Ames Research Center
Mechanical forces are potent modulators of stem cell based tissue regenerative mechanisms, inducing cell fate decisions and tissue specific commitment. A unique platform for investigating mechanotransduction is spaceflight, where microgravity and altered fluid mechanics provide a loading-null experimental condition. Seminal investigations of regenerative capacity in a wholly regenerative species, the newt model, and in a variety of totipotent and adult stem cell populations have demonstrated the detrimental effects of unloading on maintenance of stem cell based regeneration. Of particular interest is the observation that unloading interferes with the transition of stem cell pools from proliferative state to differentiation commitment. In this work we sought to test the hypothesis that gravity mechanotransduction regulates stem cell tissue regenerative processes by modulating stem cell proliferation and differentiation fates at specific cell cycle stages. To do this, clonally-derived ESCs were plated on a collagen matrix and expanded for 36 hours before re-plating on a non-adherent culture dish in the absence of leukemia inhibitory factor (LIF) to form spheroid aggregate EBs. After formation, the EBs were transferred to a collagen matrix coated culture dishes and given 4 days to allow implantation and outgrowth. In parallel, totipotent ESCs were plated 24 hours before mechanical stimulation on collagen matrix culture dishes in the presence of LIF to maintain totipotency and serve as un-differentiation committed controls. The EBs and ESCs were then subjected to either a 60 minute pulse of gravity (static loading) or 60 minutes of cyclic stretch (dynamic loading) mechanotransduction. Six hours post-stimulation, we used a 10X Genomics Single Cell controller to generate bar-coded single cell Illumina libraries and sequenced expressomes for 5,000 static loaded cells, representative of a change in gravity mechanotransduction, 5,000 dynamic loaded cells, representative of tissue loading associate with physiologic function, and 5,000 unstimulated 1g control cells. The comparison of these 3 libraries by cluster assignment based on like gene expression patterns show substantial alteration in cluster geometry due to mechanical loading. Specifically the mechanically loaded EB outgrowth cells to retain potency markers (PAX6, SOX2, CD34) and suppress early commitment markers (Dhh, VCAN, Igf1). Whereas the EBs cultured under the non-stimulated conditions display clear departure from the ESC expressome with lineage commitment markers upregulated and several tissue specific markers being expressed (BMP – early musculoskeletal development, Mesp1 – early cardiovascular cell lineage). These markers are not seen in the mechano-stimulated cultures or the totipotent ESC cultures. Comparison of like clusters between our experimental conditions revealed an array of regenerative and stem cell genes are significantly mechano-regulated. Of particular importance CDKN1a/p21, a gene shown by previous investigation of our research team to be significantly upregulated in unloading, was suppressed in the static and dynamic loaded EBS. In addition to CDKN1a/p21 many genes related to cell cycle and transitory differentiation markers had elevated expression in the mechano-stimulated EBs, but surprisingly these trends were not observed in the ESC cultures. This study is the first of its kind investigating for mechano-signaling and mechano-regulated pathways, and has already provided evidence confirming that CDKN1a/p21 expression is mechano-regulated providing a hypothetical lynchpin for stem cell transition from proliferative to differentiative states.
Enrique Sosa, Ph.D.
Molecular Cell & Developmental Biology,
Xenotransplantation and homologous transplantation of rhesus macaque primordial germ cell-like cells (rPGCLCs), generated from induced pluripotent stem cells (iPSCs), into the adult testicular niche leads to the in vivo advancement of rPGCLC differentiation. Recently, human oogonia-like cells were generated in vitro using hPGCLCs when reconstituted with female somatic cells from dissociated embryonic mouse ovaries (xrOvaries) suggesting that an embryonic niche may be required to support coordinated PGCLC differentiation. To address this, we evaluated rhesus and human PGCLC differentiation in xrTestis self-assembled from single cell suspensions of E12.5 embryonic mouse testicular cells. These single cell suspensions were aggregated as floating cultures in low adhesion 96-well plates before transferring to transwell membranes to create self-assembling rTestes. Using immunofluorescence (IF) staining, we found Sox9-positive (+) sertoli cells cluster and polarize in the rTestes, forming tubule-like structures, as early as day (D) 14 after transfer to the membrane. By D21 of transfer, these tubule-like structures become more numerous and morphologically complex. At both D14 and D21 we discovered that the extracellular matrix protein Laminin formed a basement membrane enclosing the sertoli cells as epithelial tubes. To evaluate whether this self-assembling embryonic testicular niche supports rPGCLC differentiation, we combined FACS-sorted GFP+/EpCAM+/ITAG6+ D4 PGCLCs (human or rhesus) with SSEA1 MACS-depleted embryonic mouse testicular cells (lacking endogenous mouse PGCs) to generate rhesus-mouse and human-mouse xenogeneic reconstituted testis (xrTestis). Using this approach, we observed donor-specific incorporation and survival of GFP+ PGCLCs in developing xrTestis. Taken together our findings suggest that the xrTestis may be a powerful model to study testicular niche development and physiology in vitro, as well as provide a new research tool for studying human prenatal germ cell differentiation towards in vitro gametogenesis.
Gunes Uzer, Ph.D.
Department of Mechanical and Biomedical Engineering,
Boise State University
Sensation of the mechanical qualities of the environment is critical in directing cellular function and, in the case of stem cells, regulating lineage selection. This seminar will focus on the identification of mechanical factors regulating mesenchymal stem cells in the bone marrow that provide regenerative capacity by replacing and reinforcing the skeleton at load bearing sites. The ability of mesenchymal stem cells to respond to mechanical cues generated during functional loading is critical for this capacity. The importance of the physical connections between the nucleus and the cellular cytoskeleton has become increasingly apparent in recent years as the loss of nuclear integrity underlies the etiology of clinically significant premature aging and muscle-wasting disorders. I will discuss my laboratory’s findings that how mechanical coupling of the nucleus with the cytoskeleton contributes to mesenchymal stem cell mechanosensitivity and fate selection. During the talk, I will further highlight major research themes we are pursuing towards understanding cellular and tissue level mechanical adaptations, as well as strategies for treatment and rehabilitation of musculoskeletal impairments at the cellular level.
Dampening the peripheral immune response to traumatic brain injury through intravenous delivery of umbilical cord blood stem cells in rats
Andrew Crane, Ph.D.
Department of Neurosurgery
University of Minnesota
In the United States alone, traumatic brain injury (TBI) accounts for roughly 2.8 million emergency room visits, hospitalizations, and deaths each year. Following an impact or blast injury to the skull, primary cell death at the site of injury will occur followed by a cascade of events leading to secondary cell death to brain regions outside of the impact site. Our lab previously identified a non-hematopoietic lineage of umbilical cord blood stem cells (UCBSCs) which, when delivered intravenously in rodent models of cerebral ischemia, ameliorate neurological deficits and reduced secondary cell death. Further analysis of the infiltrating immune cells in treated animals demonstrated a reduction in pro-inflammatory cells within the brain five days after treatment (seven days following ischemia). Given that stroke and TBI are characterized by neuroinflammation, we investigated the use of UCBSCs in the controlled cortical impact rodent model of TBI. Two days following unilateral impact to the primary and secondary motor cortices, rats were administered UCBSCs or vehicle via femoral vein. Seven days following UCBSC treatment, flow cytometry of mononuclear cells within the injured rat brain revealed a decrease in the number of neutrophils and an increase in the number of T lymphocytes, relative to vehicle-treated injured rats. We are continuing to explore the mechanisms by which UCBSCs exert their immunomodulatory effects, including investigating the biodistribution of the UCBSCs and the long-term benefits of stem cell therapy.
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