Recent Lineage Tracing Studies Research Articles

Mouse developmental defects, but not paraganglioma tumorigenesis, upon conditional Complex II loss in early Sox10+ cells

AbstractIn humans, loss of heterozygosity for defective alleles of any of the four subunits of mitochondrial tricarboxylic acid cycle enzyme succinate dehydrogenase (SDH, also Complex II of the electron transport chain) can lead to paraganglioma tumors in neuroendocrine cells. With the goal of developing mouse models of this rare disorder, we have developed various SDH conditional loss strategies. Based on recent lineage tracing studies, we hypothesized that conditional SDHC loss in early embryogenesis during migration of primordial neural crest cells that form the susceptible chromaffin cells of the adrenal medulla might induce paraganglioma. We triggered low levels of detectable SDHC loss in Sox10+ cells at E11.5 of mouse development. We report that, rather than developing adrenal medulla paraganglioma (pheochromocytoma), offspring survived with evidence of neural crest cell dysfunction. Phenotypes included mild lower extremity gait anomalies suggestive of neural tube closure defects and patches of unpigmented fur consistent with neural crest‐derived melanocyte dysfunction. These defects were not observed in mice lacking Sdhc knockout. Our results add to existing data suggesting that, unlike humans, even early embryonic (Sox10‐driven) SDHx loss is inadequate to trigger paraganglioma in mice of the genetic backgrounds that have been investigated. Instead, low levels of tricarboxylic acid cycle‐deficient neural crest cells cause mild developmental defects in hind limb and melanocyte function. This new model may be of interest for studies of metabolism during early neural crest cell development.

Abstract 217: Protease Activated Receptor 2 Is Critical For Vascular Smooth Muscle Cell Transition To A Macrophage-like State

Objective: Recent lineage tracing studies have shown vascular smooth muscle cells (VSMCs) develop macrophage-like characteristics (MLCs) in late-stage atherosclerosis resulting in accelerated inflammation and disease severity. Our objective was to determine if the inflammatory receptor, protease activated receptor 2 (PAR2), accelerates VSMC-MLC in a mouse model of atherosclerosis. Methods and Results: Our lab demonstrated Par2 -/- mice have decreased atherosclerotic lesion area versus proficient controls via downregulation of VSMC migration and cytokine release. To examine the role of PAR2 in VSMC dedifferentiation, Par2 +/+ and Par2 -/- VSMCs were treated with 10 ug/mL methyl- β-cyclodextrin cholesterol for 72 hours. While Par2 +/+ VSMCs demonstrated a loss of VSMCs markers (α - actin and myosin heavy chain) and upregulated macrophage markers (CD68 and Mac-2), Par2 -/- VSMCs remained stable. Looking at macrophage characteristics, Par2 -/- VSMCs phagocytized significantly less latex beads than Par2 +/+ VSMCs. Analysis of a hybrid mouse diversity panel (HMDP), conducted on 101 strains of mice with induced atherosclerosis, determined PAR2 was significantly correlated with the MLC super-gene Krüppel-like factor 4 (KLF4), which was found to have lower genetic expression in Par2 deficient VSMCs. Previous studies have demonstrated Par2 mRNA is bound and stabilized by the RNA binding protein human antigen R (HuR). Our studies confirm these findings where Par2 -/- VSMCs have less HuR mRNA expression and activity, suggesting a role of HuR in PAR2 mRNA stability. Par2 flox/flox mice bred with transgelin (SM22-alpha) Cre and low-density lipoprotein receptor deficient mice l have been establishing in our lab to investigate the effects of cell-specific deletions of PAR2 in atherosclerosis. VSMC-specific Par2 deficiency blunts the formation of atherosclerosis versus the Cre + littermates. Conclusions: These results suggest that VSMC-specific PAR2 activation mediates the dedifferentiation of VSMCs via upregulation of KLF4 and subsequent binding by HuR, which stabilizes PAR2 mRNA and upregulates PAR2. Future studies will continue to study VSMC dedifferentiation utilizing tagged lineage-tracing mouse models and various in vitro techniques.

Endothelial-to-Mesenchymal Transition in Atherosclerosis: Friend or Foe?

Despite many decades of research, complications of atherosclerosis resulting from the rupture or erosion of unstable plaques remain the leading cause of death worldwide. Advances in cellular lineage tracing techniques have allowed researchers to begin investigating the role of individual cell types in the key processes regulating plaque stability, including maintenance of the fibrous cap, a protective collagen-rich structure that underlies the endothelium. This structure was previously thought to be entirely derived from smooth muscle cells (SMC), which migrated from the vessel wall. However, recent lineage tracing studies have identified endothelial cells (EC) as an essential component of this protective barrier through an endothelial-to-mesenchymal transition (EndoMT), a process that has previously been implicated in pulmonary, cardiac, and kidney fibrosis. Although the presence of EndoMT in atherosclerotic plaques has been shown by several laboratories using EC-lineage tracing mouse models, whether EndoMT is detrimental (i.e., worsening disease progression) or beneficial (i.e., an athero-protective response that prevents plaque instability) remains uncertain as there are data to support both possibilities, which will be further discussed in this review.

HSC-independent definitive lymphopoiesis persists into adult life

Abstract The stem cell theory that all blood cells are derived from hematopoietic stem cell (HSC) is a central dogma in hematology. However, exceptions are the fetal hematopoiesis in which primitive and definitive type of erythromyeloid cells are already produced from hemogenic endothelial cells (HECs) before the first HSC emergence in the mouse embryo. This early blood cell production prior to HSC emergence is called HSC-independent hematopoiesis and considered to be transient during fetus. Recent lineage-tracing studies revealed that some tissue-resident innate immune cells (e.g. brain microglia, mast cells, and certain types of γδT cells) are also produced independently of HSCs from early extra-embryonic yolk-sac (YS). Definitive T&B-lymphoid potential and multi-potent progenitor (MPP) potential have been detected in the YS and/or embryos in in vitro studies, however, the physiological presence of these cells in the embryo at pre-HSC stage remains unknown. In addition, whether HSCs in the fetal liver are the main source of innate-like B-1a cells has been controversial. Here, using complementary lineage tracing mouse models, we show that HSC-independent MPPs and HSC-independent adoptive T & B-lymphocytes persist into adult life and are gradually replaced by HSC-derived cells. Furthermore, we found that HSCs minimally contribute to the peritoneal B-1a cells, and most B-1a cells are originated directly from ECs in the YS and embryo and HSC-independent for life. Our discovery of extensive HSC-independent MPP and adoptive lymphocytes in adults attests to the complex blood developmental dynamics through embryo to adult that underpin the immune system and challenges the paradigm of HSC theory in hematology. Supported by grant from NIH R01AI121197.

3236 – HSC-INDEPENDENT ADAPTIVE LYMPHOPOIESIS PERSISTS INTO ADULT LIFE

The stem cell theory that all blood cells are derived from hematopoietic stem cells (HSC) is a central dogma in hematology. However, exceptions are the fetal hematopoiesis in which primitive and definitive types of erythro-myeloid cells are already produced from hemogenic endothelial cells (HECs) before the first HSC emergence in the mouse embryo. This early blood cell production prior to HSC emergence is called HSC-independent hematopoiesis and is considered to be transient during the fetus. Recent lineage-tracing studies revealed that some tissue-resident innate immune cells (e.g. brain microglia, mast cells, and certain types of γδT cells) are also produced independently of HSCs from early extra-embryonic yolk-sac (YS). Definitive T&B-lymphoid potential and multi-potent progenitor (MPP) potential have been detected in the YS and/or embryos in in vitro studies, however, the physiological presence of these cells in the embryo at the pre-HSC stage remains unknown. In addition, whether HSCs in the fetal liver are the main source of innate-like B-1a cells has been controversial. Here, using complementary lineage tracing mouse models, we show that HSC-independent MPPs and HSC-independent adaptive T & B-lymphocytes persist into adult life and are gradually replaced by HSC-derived cells. Furthermore, we found that HSCs minimally contribute to the peritoneal B-1a cells, and most B-1a cells are originated directly from ECs in the YS and embryo and HSC-independent for life. Our discovery of extensive HSC-independent MPP and adaptive lymphocytes in adults attests to the complex blood developmental dynamics through embryo to adult that underpins the immune system and challenges the paradigm of HSC theory in hematology. The stem cell theory that all blood cells are derived from hematopoietic stem cells (HSC) is a central dogma in hematology. However, exceptions are the fetal hematopoiesis in which primitive and definitive types of erythro-myeloid cells are already produced from hemogenic endothelial cells (HECs) before the first HSC emergence in the mouse embryo. This early blood cell production prior to HSC emergence is called HSC-independent hematopoiesis and is considered to be transient during the fetus. Recent lineage-tracing studies revealed that some tissue-resident innate immune cells (e.g. brain microglia, mast cells, and certain types of γδT cells) are also produced independently of HSCs from early extra-embryonic yolk-sac (YS). Definitive T&B-lymphoid potential and multi-potent progenitor (MPP) potential have been detected in the YS and/or embryos in in vitro studies, however, the physiological presence of these cells in the embryo at the pre-HSC stage remains unknown. In addition, whether HSCs in the fetal liver are the main source of innate-like B-1a cells has been controversial. Here, using complementary lineage tracing mouse models, we show that HSC-independent MPPs and HSC-independent adaptive T & B-lymphocytes persist into adult life and are gradually replaced by HSC-derived cells. Furthermore, we found that HSCs minimally contribute to the peritoneal B-1a cells, and most B-1a cells are originated directly from ECs in the YS and embryo and HSC-independent for life. Our discovery of extensive HSC-independent MPP and adaptive lymphocytes in adults attests to the complex blood developmental dynamics through embryo to adult that underpins the immune system and challenges the paradigm of HSC theory in hematology.

The Dynamics of Somatic Mutagenesis During Life in Humans.

From conception to death, human cells accumulate somatic mutations in their genomes. These mutations can contribute to the development of cancer and non-malignant diseases and have also been associated with aging. Rapid technological developments in sequencing approaches in the last few years and their application to normal tissues have greatly advanced our knowledge about the accumulation of these mutations during healthy aging. Whole genome sequencing studies have revealed that there are significant differences in mutation burden and patterns across tissues, but also that the mutation rates within tissues are surprisingly constant during adult life. In contrast, recent lineage-tracing studies based on whole-genome sequencing have shown that the rate of mutation accumulation is strongly increased early in life before birth. These early mutations, which can be shared by many cells in the body, may have a large impact on development and the origin of somatic diseases. For example, cancer driver mutations can arise early in life, decades before the detection of the malignancy. Here, we review the recent insights in mutation accumulation and mutagenic processes in normal tissues. We compare mutagenesis early and later in life and discuss how mutation rates and patterns evolve during aging. Additionally, we outline the potential impact of these mutations on development, aging and disease.

Seamless Genetic Recording of Transiently Activated Mesenchymal Gene Expression in Endothelial Cells During Cardiac Fibrosis.

Cardiac fibrosis is a lethal outcome of excessive formation of myofibroblasts that are scar-forming cells accumulated after heart injury. It has been reported that cardiac endothelial cells (ECs) contribute to a substantial portion of myofibroblasts through endothelial to mesenchymal transition (EndoMT). Recent lineage tracing studies demonstrate that myofibroblasts are derived from the expansion of resident fibroblasts rather than from the transdifferentiation of ECs. However, it remains unknown whether ECs can transdifferentiate into myofibroblasts reversibly or EndoMT genes were just transiently activated in ECs during cardiac fibrosis. By using the dual recombination technology based on Cre-loxP and Dre-rox, we generated a genetic lineage tracing system for tracking EndoMT in cardiac ECs. We used it to examine if there is transiently activated mesenchymal gene expression in ECs during cardiac fibrosis. Activation of the broadly used marker gene in myofibroblasts, αSMA (α-smooth muscle actin), and the transcription factor that induces epithelial to mesenchymal transition, Zeb1 (zinc finger E-box-binding homeobox 1), was examined. The genetic system enables continuous tracing of transcriptional activity of targeted genes in vivo. Our genetic fate mapping results revealed that a subset of cardiac ECs transiently expressed αSMA and Zeb1 during embryonic valve formation and transdifferentiated into mesenchymal cells through EndoMT. Nonetheless, they did not contribute to myofibroblasts, nor transiently expressed αSMA or Zeb1 after heart injury. Instead, expression of αSMA was activated in resident fibroblasts during cardiac fibrosis. Mesenchymal gene expression is activated in cardiac ECs through EndoMT in the developing heart, but ECs do not transdifferentiate into myofibroblasts, nor transiently express some known mesenchymal genes during homeostasis and fibrosis in the adult heart. Resident fibroblasts that are converted to myofibroblasts by activating mesenchymal gene expression are the major contributors to cardiac fibrosis.

Increased Plasma Levels of Myosin Heavy Chain 11 Is Associated with Atherosclerosis.

Many studies have revealed numerous potential biomarkers for atherosclerosis, but tissue-specific biomarkers are still needed. Recent lineage-tracing studies revealed that smooth muscle cells (SMCs) contribute substantially to plaque formation, and the loss of SMCs causes plaque vulnerability. We investigated the association of SMC-specific myosin heavy chain 11 (myosin-11) with atherosclerosis. Forty-five patients with atherosclerosis and 34 control subjects were recruited into our study. In the atherosclerosis patients, 35 patients had either coronary artery disease (CAD) or peripheral artery disease (PAD), and 10 had both CAD and PAD. Coronary arteries isolated from five patients were subjected to histological study. Circulating myosin-11 levels were higher in the CAD or PAD group than in controls. The area under the receiver operating characteristic curve of myosin-11 was 0.954. Circulating myosin-11 levels in the CAD and PAD group were higher than in the CAD or PAD group, while high-sensitivity C-reactive protein concentrations did not differ between these groups. Multinomial logistic regression analyses showed a significant association of myosin-11 levels with the presence of multiple atherosclerotic regions. Myosin-11 was expressed in the medial layer of human atherosclerotic lesions where apoptosis elevated. Circulating myosin-11 levels may be useful for detecting spatial expansion of atherosclerotic regions.

MiRNA-17-92 cluster is required for the embryonic development of Langerhans cells and dendritic epidermal T cells

Abstract Mouse epidermis contains two major immune cells, antigen presenting Langerhans cells (LCs) and dendritic epidermal T cells (DETCs), which regulate skin immunity and involve in disease pathogenesis. Recent lineage-tracing studies have uncovered that mouse DETCs and LCs are derived from embryonic yolk-sac-derived hematopoietic precursors and self-maintain after birth. However, detailed regulating mechanisms related to their ontogeny and homeostasis remain unclear. MicroRNAs (miRNAs) are important post transcriptional regulators of protein-coding genes. Using a CSF1R-cre-mediated Dicer deletion mouse model, we found the critical involvement of miRNAs in the ontogeny of both LCs and DETCs. To further investigate the role of individual miRNAs in the ontogeny of LCs and DETCs, we evaluated the expression of miRNAs in the precursors of LCs and DETCs at different embryonic developmental stages and identified that miR17-92 cluster was highly expressed and dynamically regulated. We next generated a Csf1rCre-mediated miR17-92 deletion mouse model (miR17-92 KO) and found that conditionally deletion of miR17-92 led to significantly reduced number and interrupted phenotypes of epidermis LC and DETC precursors at E18.5 and P0 of miR17-92 KO embryos. Defective LC precursors were also identified at E14.5 and E16.5 embryonic skin, while diminished number and blocked maturation of Vg3+ DETC precursors were identified in the thymus of the same embryonic stages in miR17-92 KO mice. Overall, our results indicate that miR17-92 serves as a key epigenetic regulator in the ontogeny of LCs and DETCs. Detailed molecular mechanisms underlining the miR17-92 mediated LC and DETC developmental regulation are currently under investigation.

Systems analysis of hematopoiesis using single-cell lineage tracing

In the last few decades, revolutionary advances in next-generation sequencing have led to single-cell lineage tracing technologies that now enable researchers to identify and quantify hematopoietic cell behavior with unprecedented detail. Combined readouts of cell lineage and cell state from the same cell mitigate the need to prospectively isolate populations of interest, and allow a system-level understanding of dynamic developmental processes. We will discuss the advantages and shortcomings of these technologies, the intriguing discoveries that stemmed from lineage tracing hematopoiesis at the single-cell level and the directions toward which the field is moving. Single-cell lineage tracing studies unveiled extensive functional heterogeneity within discrete immunophenotypic populations. Recently, several groups merged lineage tracing with single-cell RNA sequencing to visualize clonal relationships directly on transcriptional landscapes without the requirement for prospective isolation of cell types by FACS. To study the cell dynamics of hematopoiesis, without perturbation in their native niche, researchers have developed mouse models with endogenous single-cell lineage tracing systems, which can simultaneously trace thousands of hematopoietic progenitor cells in a single mouse, without transplantation. The emerging picture is that multiple hematopoietic hierarchies coexist within a single individual, each with distinct regulatory features. These hierarchies are imprinted during development much earlier than previously predicted, persisting well into adulthood and even after injury and transplantation. Clone-tracking experiments allow stem-cell researchers to characterize lineage hierarchies during blood development and regeneration. Combined with single-cell genomics analyses, these studies are allowing system-level description of hematopoiesis in mice and humans. Early exploratory studies have unveiled features with important implications for human biology and disease. VIDEO ABSTRACT.

Cellular Origins of Beige Fat Cells Revisited.

Activated beige adipocytes have therapeutic potential due to their ability to improve glucose and lipid homeostasis. To date, the origin of beige adipocytes remains enigmatic. Whether beige cells arise through de novo differentiation from resident precursors or through reprogramming of mature white adipocytes has been a topic of intense discussion. Here, we offer our perspective on the natural origin of beige adipocytes in mice. In particular, we revisit recent lineage-tracing studies that shed light on this issue and offer new insight into how environmental housing temperatures early in life influence the mode of beige adipocyte biogenesis upon cold exposure later in life. We suggest a unified model in which beige adipocytes (UCP1+ multilocular cells) in rodents initially arise predominantly from progenitors (i.e., de novo beige adipogenesis) upon the first exposure to cold temperatures and then interconvert between "dormant beige" and "active beige" phenotypes (i.e., beige cell activation) upon subsequent changes in environmental temperature. Importantly, we highlight experimental considerations needed to visualize de novo adipogenesis versus beige cell activation in mice. A precise understanding of the cellular origins of beige adipocytes emanating in response to physiological and pharmacological stimuli may better inform therapeutic strategies to recruit beige adipocytes in vivo.

Deciphering White Adipose Tissue Heterogeneity.

Adipose tissue not only stores energy, but also controls metabolism through secretion of hormones, cytokines, proteins, and microRNAs that affect the function of cells and tissues throughout the body. Adipose tissue is organized into discrete depots throughout the body, and these depots are differentially associated with insulin resistance and increased risk of metabolic disease. In addition to energy-dissipating brown and beige adipocytes, recent lineage tracing studies have demonstrated that individual adipose depots are composed of white adipocytes that are derived from distinct precursor populations, giving rise to distinct subpopulations of energy-storing white adipocytes. In this review, we discuss this developmental and functional heterogeneity of white adipocytes both between and within adipose depots. In particular, we will highlight findings from our recent manuscript in which we find and characterize three major subtypes of white adipocytes. We will discuss these data relating to the differences between subcutaneous and visceral white adipose tissue and in relationship to previous work deciphering adipocyte heterogeneity within adipose tissue depots. Finally, we will discuss the possible implications of adipocyte heterogeneity may have for the understanding of lipodystrophies.

Long-Term Engraftment of ESC-Derived B-1 Progenitor Cells Supports HSC-Independent Lymphopoiesis.

SummaryIt is generally considered that mouse embryonic stem cell (ESC) differentiation into blood cells in vitro recapitulates yolk sac (YS) hematopoiesis. As such, similar to YS-derived B-progenitors, we demonstrate here that ESC-derived B-progenitors differentiate into B-1 and marginal zone B cells, but not B-2 cells in immunodeficient mice after transplantation. ESC-derived B-1 cells were maintained in the recipients for more than 6 months, secreting natural IgM antibodies in vivo. Gene expression profiling displayed a close relationship between ESC- and YS-derived B-1 progenitors. Because there are no hematopoietic stem cells (HSCs) detectable in our ESC differentiation culture, successful long-term engraftment of ESC-derived functional B-1 cells supports the presence of HSC-independent B-1 cell development.

B-1 cell responses to infections.

B-1 cells represent an innate-like early-developing B cell population, whose existence as an independent lymphocyte subset has been questioned in the past. Recent molecular and lineage tracing studies have not only confirmed their unique origins and differentiation paths, they have also provided a rationale for their distinctive functionalities compared to conventional B cells. This review summarizes our current understanding of B-1 cell development, and the activation events that regulate B-1 cell responses to self and foreign antigens. We discuss the unresolved question to what extent BCR engagement, that is, antigen-specificity versus innate signaling contributes to B-1 cell's participation in tissue homeostasis and immune defense as providers of 'natural' and antigen-induced antibody responses, and as cytokine-producing immune regulators.

Cellular Plasticity of Mammary Epithelial Cells Underlies Heterogeneity of Breast Cancer.

The hierarchical relationships between stem cells, lineage-committed progenitors, and differentiated cells remain unclear in several tissues, due to a high degree of cell plasticity, allowing cells to switch between different cell states. The mouse mammary gland, similarly to other tissues such as the prostate, the sweat gland, and the respiratory tract airways, consists of an epithelium exclusively maintained by unipotent progenitors throughout adulthood. Such unipotent progenitors, however, retain a remarkable cellular plasticity, as they can revert to multipotency during epithelial regeneration as well as upon oncogene activation. Here, we revise the current knowledge on mammary cell hierarchies in light of the most recent lineage tracing studies performed in the mammary gland and highlight how stem cell differentiation or reversion to multipotency are at the base of tumor development and progression. In addition, we will discuss the current knowledge about the interplay between tumor cells of origin and defined genetic mutations, leading to different tumor types, and its implications in choosing specific therapeutic protocols for breast cancer patients.

Clonal analysis of Notch1-expressing cells reveals the existence of unipotent stem cells that retain long-term plasticity in the embryonic mammary gland

Recent lineage tracing studies have revealed that mammary gland homeostasis relies on unipotent stem cells. However, whether and when lineage restriction occurs during embryonic mammary development, and which signals orchestrate cell fate specification, remain unknown. Using a combination of in vivo clonal analysis with whole mount immunofluorescence and mathematical modelling of clonal dynamics, we found that embryonic multipotent mammary cells become lineage-restricted surprisingly early in development, with evidence for unipotency as early as E12.5 and no statistically discernable bipotency after E15.5. To gain insights into the mechanisms governing the switch from multipotency to unipotency, we used gain-of-function Notch1 mice and demonstrated that Notch activation cell autonomously dictates luminal cell fate specification to both embryonic and basally committed mammary cells. These functional studies have important implications for understanding the signals underlying cell plasticity and serve to clarify how reactivation of embryonic programs in adult cells can lead to cancer.

"Attack of the Clones": Commonalities Between Cancer and Atherosclerosis.

Many studies have noted similarities between atherosclerosis and cancer including pronounced cellular plasticity, clonal expansion of cellular subtypes, increased DNA mutations, defective efferocytosis pathways, and an important role for proto-oncogenes in disease development. Although it is clear that these 2 diseases have disparate causes, noting the parallels between atherosclerosis and cancer may help us identify unique, targeted therapeutic strategies. Early articles described atheromas as benign neoplasms of the blood vessel comprised primarily of fibrous smooth muscle cells (SMCs). However, additional studies soon noted the presence of macrophages and other cell types, thereby promoting controversy as to which cells actually give rise to the atherosclerotic plaque. Much of this controversy results from the promiscuity of the so-called lineage-specific markers.1 For example, bone marrow–derived cells can migrate into plaques and begin to express certain SMC markers,2 whereas SMCs can upregulate macrophage-specific markers such as galectin-3, CD11b, and F4/80 as they migrate into the plaque.3,4 In addition, recent lineage tracing studies suggest that endothelial cells can acquire mesenchymal cell characteristics, and some adventitial cells may have the capability of forming SMC-like cells. Collectively, these studies demonstrate the impressive capacity of many vascular cell types to dedifferentiate or transdifferentiate in response to the atherosclerotic environment and suggest that plaques may arise from multiple vascular and circulating cell types. Clearly, cellular plasticity is a major theme in atherosclerotic plaque development—as it is in cancer—and methods to reprogram proatherosclerotic cells could have tremendous therapeutic potential. The clonal evolution theory of cancer development posits that certain tumors arise from a single cell that expands in response to a series of acquired mutations. In 1973, Benditt and Benditt5 performed X-inactivation studies on human atherosclerotic lesions and found evidence that atherosclerotic plaques could be derived from a single cell that underwent monoclonal expansion, …

Conversion of Terminally Committed Hepatocytes to Culturable Bipotent Progenitor Cells with Regenerative Capacity

A challenge for advancing approaches to liver regeneration is loss of functional differentiation capacity when hepatocyte progenitors are maintained in culture. Recent lineage-tracing studies have shown that mature hepatocytes (MHs) convert to an immature state during chronic liver injury, and we investigated whether this conversion could be recapitulated invitro and whether such converted cells could represent a source of expandable hepatocytes. We report that a cocktail of small molecules, Y-27632, A-83-01, and CHIR99021, can convert rat and mouse MHs invitro into proliferative bipotent cells, which we term chemically induced liver progenitors (CLiPs). CLiPs can differentiate into both MHs and biliary epithelial cells that can form functional ductal structures. CLiPs in long-term culture did not lose their proliferative capacity or their hepatic differentiation ability, and rat CLiPs were shown to extensively repopulate chronically injured liver tissue. Thus, our study advances the goals of liver regenerative medicine.

Abstract 254: Platelet Derived Growth Factor Receptor Beta Activation Promotes Atheroprotective Changes in Smooth Muscle Cell Phenotype

Despite decades of research, little is known about mechanisms and factors driving atherosclerotic progression leading to plaque rupture, thrombosis and myocardial infarction or stroke. The widely accepted dogma is a thin fibrous cap and a paucity of Acta2+ smooth muscle cells (SMC) relative to macrophages increases risk of plaque rupture. However, our recent rigorous lineage tracing studies in advanced lesions showed a subset of cells that express macrophage markers are SMC-derived and SMC specific conditional knockout of stem pluripotency gene Klf4 resulted in increased fibrous cap thickness and decreased lesion size and abundance of SMC-derived macrophage-like cells. Results indicate that SMC play a critical role in lesion pathogenesis but paradoxically can transition to a beneficial or detrimental state, presumably as a function of the local environmental milieu within lesions. Studies highlight the critical need to identify factors and mechanisms that direct SMC to form a stable fibrous cap. Various in vitro data show that SMC treated with platelet derived growth factor receptor beta (PDGFR beta) ligands dedifferentiate, proliferate, and synthesize extracellular matrix (ECM) molecules, including collagens known to be enriched within the fibrous cap. We hypothesize that PDGFR beta activation in SMC is necessary for fibrous cap stabilization through EMC protein synthesis. Herein we employ Myh11-ER T2 -cre eYFP SMC lineage tracing ApoE-/- Western diet fed mice to show that a subset of SMC in advanced lesions express PDGFR beta and are localized to the fibrous cap. Moreover we demonstrate that SMC specific conditional KO of the PDGFR beta in these lineage tracing mice resulted in dramatic reductions in the numbers of YFP+ (SMC-derived) cells within lesions but an increase in YFP- (not SMC-derived) macrophages, as well as a decrease in Acta2+ cells in the fibrous cap. Results indicate that PDGFR beta activation in SMC is a critical determinant of the cellular composition of lesions and is essential for investment of SMC within the fibrous cap. Supported by NIH R01 grants HL121008 and HL087867 to GKO.

High fat diet promotes prostatic basal-to-luminal differentiation and accelerates initiation of prostate epithelial hyperplasia originated from basal cells

Recent lineage tracing studies showed that the prostate basal and luminal cells in adult mice are two independent lineages under the physiological condition, but basal cells are capable of generating luminal progenies during bacterial infection-induced prostatitis. Because acute bacterial infection in human prostate tissues is relatively rare, the disease relevance of the bacterial infection-induced basal-to-luminal differentiation is uncertain. Herein we employ a high fat diet-induced sterile prostate inflammation model to determine whether basal-to-luminal differentiation can be induced by inflammation irrespective of the underlying etiologies. A K14-CreER model and a fluorescent report line are utilized to specifically label basal cells with the green fluorescent protein. We show that high fat diet promotes immune cell infiltration into the prostate tissues and basal-to-luminal differentiation. Increased cell proliferation accompanies basal-to-luminal differentiation, suggesting a concurrent regulation of basal cell proliferation and differentiation. This study demonstrates that basal-to-luminal differentiation can be induced by different types of prostate inflammation evolved with distinct etiologies. Finally, high fat diet also accelerates initiation and progression of prostatic intraepithelial neoplasia that are originated from basal cells with loss-of-function of the tumor suppressor Pten. Because prostate cancer originated from basal cells tends to be invasive, our study also provides an alternative explanation for the association between obesity and aggressive prostate cancer.