Multilineage Progenitor Cells Research Articles

Stem Cell Administration to Repair Torn Menisci

The meniscus is a crescent-shaped, fibrocartilaginous padding in the knee that contains variable vascularization and histology. This structure enables proper loadbearing, knee movement, and protection of articular cartilage and bone of the tibiofemoral joint. The meniscus is subject to tears from acute injury and degenerative stresses. Many meniscal tears do not heal naturally due to poor vascularization and high stresses placed on the meniscus. These often result in pain and mechanical symptoms and are highly correlated to osteoarthritis (OA) development. While there are a wide variety of suggested treatments for meniscal tears, studies indicate common surgical interventions have little to no significant improvements in abating patient symptoms or limiting osteoarthritic progression, with high failure rates. Therefore, alternative treatments are being actively explored for meniscal tear repair. One of the most-researched treatment options is using mesenchymal stem cells (MSC), a type of adult multilineage progenitor cell capable of facilitating meniscal tissue regeneration. Many different types of MSCs and supplemental, cuttingedge techniques are being tested to maximize tear healing. MSCs hold great promise for regenerating meniscal tissue, limiting OA, and restoring joint functionality. However, more research is required to prove the significance of stem cell treatments in humans and define the conditions for their use.

Identification of the natural chalcone glycoside hydroxysafflor yellow A as a suppressor of P53 overactivation-associated hematopoietic defects.

Enhanced P53 signaling may lead to hematopoietic disorders, yet an effective therapeutic strategy is still lacking. Our study, along with previous research, suggests that P53 overactivation and hematopoietic defects are major consequences of zinc deficiency. However, the relationship between these two pathological processes remains unclear. In this study, we observed a severe reduction in the number of hematopoietic stem cells (HSCs) and multi-lineage progenitor cells in zebrafish treated with the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine and showed the indispensable role of P53 signaling in the process. Next, we took advantage of HSCs-labeled transgenic zebrafish and conducted a highly efficient phenotypic screening for small molecules against P53-dependent hematopoietic disorders. Hydroxysafflor yellow A (HSYA), a natural chalcone glycoside, exhibited potent protection against hematopoietic failure in zinc-deficient zebrafish and strongly inhibited the P53 pathway. We confirmed the protective effect of HSYA in zinc-deficient mice bone marrow nucleated cells, which showed a significant suppression of P53 signaling and oxidative stress. Furthermore, the hematopoietic-protective activity of HSYA was validated using a mice model of myelotoxicity induced by 5-FU. In summary, our work provides an effective phenotypic screening strategy for identifying hematopoietic-protective agents and reveals the novel role of HSYA as a promising lead compound in rescuing hematopoietic disorders associated with P53 overactivation.

Lateral thinking in syndromic congenital cardiovascular disease.

Syndromic birth defects are rare diseases that can present with seemingly pleiotropic comorbidities. Prime examples are rare congenital heart and cardiovascular anomalies that can be accompanied by forelimb defects, kidney disorders and more. Whether such multi-organ defects share a developmental link remains a key question with relevance to the diagnosis, therapeutic intervention and long-term care of affected patients. The heart, endothelial and blood lineages develop together from the lateral plate mesoderm (LPM), which also harbors the progenitor cells for limb connective tissue, kidneys, mesothelia and smooth muscle. This developmental plasticity of the LPM, which founds on multi-lineage progenitor cells and shared transcription factor expression across different descendant lineages, has the potential to explain the seemingly disparate syndromic defects in rare congenital diseases. Combining patient genome-sequencing data with model organism studies has already provided a wealth of insights into complex LPM-associated birth defects, such as heart-hand syndromes. Here, we summarize developmental and known disease-causing mechanisms in early LPM patterning, address how defects in these processes drive multi-organ comorbidities, and outline how several cardiovascular and hematopoietic birth defects with complex comorbidities may be LPM-associated diseases. We also discuss strategies to integrate patient sequencing, data-aggregating resources and model organism studies to mechanistically decode congenital defects, including potentially LPM-associated orphan diseases. Eventually, linking complex congenital phenotypes to a common LPM origin provides a framework to discover developmental mechanisms and to anticipate comorbidities in congenital diseases affecting the cardiovascular system and beyond.

Linking Benzene, in Utero Carcinogenicity and Fetal Hematopoietic Stem Cell Niches: A Mechanistic Review.

Previous research reported that prolonged benzene exposure during in utero fetal development causes greater fetal abnormalities than in adult-stage exposure. This phenomenon increases the risk for disease development at the fetal stage, particularly carcinogenesis, which is mainly associated with hematological malignancies. Benzene has been reported to potentially act via multiple modes of action that target the hematopoietic stem cell (HSCs) niche, a complex microenvironment in which HSCs and multilineage hematopoietic stem and progenitor cells (HSPCs) reside. Oxidative stress, chromosomal aberration and epigenetic modification are among the known mechanisms mediating benzene-induced genetic and epigenetic modification in fetal stem cells leading to in utero carcinogenesis. Hence, it is crucial to monitor exposure to carcinogenic benzene via environmental, occupational or lifestyle factors among pregnant women. Benzene is a well-known cause of adult leukemia. However, proof of benzene involvement with childhood leukemia remains scarce despite previously reported research linking incidences of hematological disorders and maternal benzene exposure. Furthermore, accumulating evidence has shown that maternal benzene exposure is able to alter the developmental and functional properties of HSPCs, leading to hematological disorders in fetus and children. Since HSPCs are parental blood cells that regulate hematopoiesis during the fetal and adult stages, benzene exposure that targets HSPCs may induce damage to the population and trigger the development of hematological diseases. Therefore, the mechanism of in utero carcinogenicity by benzene in targeting fetal HSPCs is the primary focus of this review.

Periodontal Tissue Regeneration by Transplantation of Autologous Adipose Tissue-Derived Multi-Lineage Progenitor Cells With Carbonate Apatite

We have developed an autologous transplantation method using adipose tissue-derived multi-lineage progenitor cells (ADMPCs) as a method of periodontal tissue regeneration that can be adapted to severe periodontal disease. Our previous clinical study confirmed the safety of autologous transplantation of ADMPCs and demonstrated its usefulness in the treatment of severe periodontal disease. However, in the same clinical study, we found that the fibrin gel used as the scaffold material might have caused gingival recession and impaired tissue regeneration in some patients. Carbonate apatite has a high space-making capacity and has been approved in Japan for periodontal tissue regeneration. In this study, we selected carbonate apatite as a candidate scaffold material for ADMPCs and conducted an in vitro examination of its effect on the cellular function of ADMPCs. We further performed autologous ADMPC transplantation with carbonate apatite as the scaffold material in a model of one-wall bone defects in beagles and then analyzed the effect on periodontal tissue regeneration. The findings showed that carbonate apatite did not affect the cell morphology of ADMPCs and that it promoted proliferation. Moreover, no effect on secretor factor transcription was found. The results of the in vivo analysis confirmed the space-making capacity of carbonate apatite, and the acquisition of significant new attachment was observed in the group involving ADMPC transplantation with carbonate apatite compared with the group involving carbonate apatite application alone. Our results demonstrate the usefulness of carbonate apatite as a scaffold material for ADMPC transplantation.

Deep learning predicts the differentiation of kidney organoids derived from human induced pluripotent stem cells.

Kidney organoids derived from human pluripotent stem cells (hPSCs) contain multilineage nephrogenic progenitor cells and can recapitulate the development of the kidney. Kidney organoids derived from hPSCs have the potential to be applied in regenerative medicine as well as renal disease modeling, drug screening, and nephrotoxicity testing. Despite biotechnological advances, individual differences in morphological and growth characteristics among kidney organoids need to be addressed before clinical and commercial application. In this study, we hypothesized that an automated noninvasive method based on deep learning of bright-field images of kidney organoids can predict their differentiation status. Bright-field images of kidney organoids were collected on day 18 after differentiation. To train convolutional neural networks (CNNs), we utilized a transfer learning approach. CNNs were trained to predict the differentiation of kidney organoids on bright-field images based on the messenger RNA expression of renal tubular epithelial cells as well as podocytes. The best prediction model was DenseNet121 with a total Pearson correlation coefficient score of 0.783 on a test dataset. W classified the kidney organoids into two categories: organoids with above-average gene expression (Positive) and those with below-average gene expression (Negative). Comparing the best-performing CNN with human-based classifiers, the CNN algorithm had a receiver operating characteristic-area under the curve (AUC) score of 0.85, while the experts had an AUC score of 0.48. These results confirmed our original hypothesis and demonstrated that our artificial intelligence algorithm can successfully recognize the differentiation status of kidney organoids.

Periodontal tissue regeneration by transplantation of autologous adipose tissue-derived multi-lineage progenitor cells

Periodontitis is a chronic inflammatory disease that destroys tooth-supporting periodontal tissue. Current periodontal regenerative therapies have unsatisfactory efficacy; therefore, periodontal tissue engineering might be established by developing new cell-based therapies. In this study, we evaluated the safety and efficacy of adipose tissue-derived multi-lineage progenitor cells (ADMPC) autologous transplantation for periodontal tissue regeneration in humans. We conducted an open-label, single-arm exploratory phase I clinical study in which 12 periodontitis patients were transplanted with autologous ADMPCs isolated from subcutaneous adipose tissue. Each patient underwent flap surgery during which autologous ADMPCs were transplanted into the bone defect with a fibrin carrier material. Up to 36 weeks after transplantation, we performed a variety of clinical examinations including periodontal tissue inspection and standardized dental radiographic analysis. A 36-week follow-up demonstrated no severe transplantation-related adverse events in any cases. ADMPC transplantation reduced the probing pocket depth, improved the clinical attachment level, and induced neogenesis of alveolar bone. Therapeutic efficiency was observed in 2- or 3-walled vertical bone defects as well as more severe periodontal bone defects. These results suggest that autologous ADMPC transplantation might be an applicable therapy for severe periodontitis by inducing periodontal regeneration.

Diabetic sarcopenia: metabolic and molecular appraisal.

Myopathy is the missing slot from the routine clinical checkup for diabetic complications. Similarly, its pathophysiological, metabolic, and molecular bases are insufficiently explored. In this review, the above issues are highlighted with a focus on skeletal muscle atrophy (also described as diabetic sarcopenia), in contrast to the normal histological, physiological, and molecular features of the muscles. Literature search using published data from different online resources was used. Several diabetic myopathy etiological factors are discussed explicitly including; inflammation and immunological responses, with emphasis on TNFα and IL-6 overproduction, oxidative stress, neuropathy and vasculopathy, aging sarcopenia, antidiabetic drugs, and insulin resistance as a denominator. The pathophysiological hallmark of diabetic muscle atrophy is the decreased muscle proteins synthesis and increased degradation. The muscle protein degradation is conveyed by 4 systems; ubiquitin-proteasome, lysosomal autophagy, caspase-3, and calpain systems, and is mostly mediated via the IL6/STAT, TNF&IL6/NFκB, myostatin/Smad2/3, and FOXO1/3 signaling pathways, while the protein synthesis inhibition is mediated via suppression of the IGF1-PI3K-Akt-mTOR, and SC-Gαi2-pathways. Moreover, the satellite cells and multilineage muscle mesenchymal progenitor cells differentiation plays a major role on the fate of the affected muscle cells by taking an adipogenic, fibrogenic, or connective tissue lineage. As a conclusion, in this article, the pathological features of diabetic sarcopenia are reviewed at gross level, while at a molecular level the normal protein turnover, signal transduction, and pathways involved in muscle atrophy are described. Finally, an integrated network describing the molecular partakers in diabetic sarcopenia is presented.

Blood Vessel Resident Human Stem Cells in Health and Disease.

The vascular wall is comprised of distinct layers controlling angiogenesis, blood flow, vessel anchorage within organs, and cell and molecule transit between blood and tissues. Moreover, some blood vessels are home to essential stem-like cells, a classic example being the existence in the embryo of hemogenic endothelial cells at the origin of definitive hematopoiesis. In recent years, microvascular pericytes and adventitial perivascular cells were observed to include multi-lineage progenitor cells involved not only in organ turnover and regeneration but also in pathologic remodeling, including fibrosis and atherosclerosis. These perivascular mesodermal elements were identified as native forerunners of mesenchymal stem cells. We have presented in this brief review our current knowledge on vessel wall-associated tissue remodeling cells with respect to discriminating phenotypes, functional diversity in health and disease, and potential therapeutic interest.

Single cell multi-omic analysis identifies a Tbx1-dependent multilineage primed population in murine cardiopharyngeal mesoderm

The poles of the heart and branchiomeric muscles of the face and neck are formed from the cardiopharyngeal mesoderm within the pharyngeal apparatus. They are disrupted in patients with 22q11.2 deletion syndrome, due to haploinsufficiency of TBX1, encoding a T-box transcription factor. Here, using single cell RNA-sequencing, we now identify a multilineage primed population within the cardiopharyngeal mesoderm, marked by Tbx1, which has bipotent properties to form cardiac and branchiomeric muscle cells. The multilineage primed cells are localized within the nascent mesoderm of the caudal lateral pharyngeal apparatus and provide a continuous source of cardiopharyngeal mesoderm progenitors. Tbx1 regulates the maturation of multilineage primed progenitor cells to cardiopharyngeal mesoderm derivatives while restricting ectopic non-mesodermal gene expression. We further show that TBX1 confers this balance of gene expression by direct and indirect regulation of enriched genes in multilineage primed progenitors and downstream pathways, partly through altering chromatin accessibility, the perturbation of which can lead to congenital defects in individuals with 22q11.2 deletion syndrome.

Differentiation of immortalized human multi-lineage progenitor to alveolar type 2-like cells: angiotensin-converting enzyme 2 expression and binding of severe acute respiratory syndrome coronavirus 2 spike and spike 1 proteins

Along with the nasal epithelium, the lung epithelium is a portal of entry for sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and many other respiratory viruses. In the case of SARS-CoV-2, the virus surface spike proteins bind to the angiotensin-converting enzyme 2 (ACE-2) receptor to facilitate entry into the respiratory epithelium. Alveolar type 2 (AT2) cells are committed respiratory progenitor cells responsible for the integrity and regeneration of the respiratory epithelium and production of respiratory surfactant proteins. AT2 cells express high levels of surface ACE-2 and thus are a leading target for primary infection by SARS-CoV-2. This study describes a method for directly differentiating telomerase reverse transcriptase-immortalized human cord blood-derived multi-lineage progenitor cells (MLPCs) to AT2-like cells for the purpose of generating an in vitro cellular platform for viral studies. Differentiation was confirmed with the acquisition of AT2 and absence of alveolar type 1 (AT1) specific markers by confocal microscopy. Expression of the ACE-2 receptor was confirmed by immunofluorescence antibody staining, quantitative reverse transcription polymerase chain reaction and binding of biotinylated SARS-CoV-2 spike and spike 1 proteins. The binding of biotinylated spike proteins was specifically blocked by unlabeled spike proteins and neutralizing antibodies. Additionally, it was demonstrated that the spike protein was internalized after binding to the surface membrane of the cells. The authors defined the culture conditions that enabled AT2-like cells to be repeatedly passaged and cryopreserved without further differentiation to AT1. The authors’ method provides a stable and renewable source of AT2 cells for respiratory viral binding, blocking and uptake studies.

Radiation-Induced Senescence in p16+/LUC Mouse Lung Compared to Bone Marrow Multilineage Hematopoietic Progenitor Cells.

We defined the time course of ionizing radiation-induced senescence in lung compared to bone marrow of p16+/LUC mice in which the senescence-induced biomarker (p16) is linked to a luciferase reporter gene. Periodic in situ imaging revealed increased luciferase activity in the lungs of 20 Gy thoracic irradiated, but not 8 Gy total-body irradiated (TBI) mice beginning at day 75 and increasing to day 170. In serial sections of explanted lungs, senescent cells appeared in the same areas as did fibrosis in the 20 Gy thoracic irradiated, but not the 8 Gy TBI group. Lungs from 8 Gy TBI mice at one year did show increased RNA levels for p16, p21, p19 and TGF-β. Individual senescent cells in 20 Gy irradiated mouse lung included those with epithelial, endothelial, fibroblast and hematopoietic cell biomarkers. Rare senescent cells in the lungs of 8 Gy TBI mice at one year were of endothelial phenotype. Long-term bone marrow cultures (LTBMCs) were established at either day 60 or one year after 8 Gy TBI. In freshly removed marrow at both times after irradiation, there were increased senescent cells. In LTBMCs, there were increased senescent cells in both weekly harvested single cells and in colonies of multilineage hematopoietic progenitor cells producing CFU-GEMM (colony forming unit-granulocyte, erythrocyte, monocyte/macrophage, mega-karyocyte) that were formed in secondary cultures when these single cells were plated in semisolid media. LTBMCs from TBI mice produced fewer CFU-GEMM; however, the relative percentage of senescent cell-containing colonies was increased as measured by both p16-luciferase and β-galactosidase. Therefore, 20 Gy thoracic radiation, as well as 8 Gy TBI, induces senescent cells in the lungs. With bone marrow, 8 Gy TBI induced senescence in both hematopoietic cells and in colony-forming progenitors. The p16+/LUC mouse strain provides a valuable system in which to compare the kinetics of radiation-induced senescence between organs in vivo, and to evaluate the potential role of senescent cells in irradiation pulmonary fibrosis.

Functional and Phenotypic Characterization of Cord Blood and Bone Marrow Subsets Expressing FLT3 (CD135) Receptor Tyrosine Kinase

AbstractThe class III receptor tyrosine kinase FLT3/FLK2 (FLT3; CD135) represents an important molecule involved in early steps of hematopoiesis. Here we compare cell-surface expression of FLT3 on bone marrow (BM) and cord blood (CB) cells using monoclonal antibodies (MoAbs) specific for the extracellular domain of human FLT3. Flow cytometric analysis of MACS-purified BM and CB cells showed that 63% to 82% of BM CD34+ and 88% to 95% of the CB CD34+ cells coexpress FLT3. Clonogenic assays and morphological characterization of FACS-sorted BM CD34+ cells demonstrate that colony-forming unit–granulocyte-macrophage (CFU-GM) and immature myelo-monocytic precursor cells are enriched in the subpopulation staining most brightly with the FLT3 MoAb whereas the majority of the burst-forming units-erythroid (BTU-E) and small cells with lymphoid morphology are found in the FLT3− population. In contrast, statistically indistinguishable proportions of CFU-granulocyte-erythrocyte-megakaryocyte-macrophage (CFU-GEMM) and more primitive cobblestone area forming cells (CAFC) were detected in both fractions, albeit the FLT3+ fraction consistently showed more CAFC activity than the FLT3− fraction. Although in both, BM and CB the majority of CD34+CD117+ (KIT+), CD34+CD90+ (Thy-1+), and CD34+CD109+ cells coexpress FLT3, three-color phenotypic analyses are consistent with the functional findings and suggest that the most primitive cells defined as CD34+CD38−, CD34+CD71low, CD34+HLA-DR−, CD34+CD117low, CD34+CD90+, and CD34+CD109+ express low levels of cell-surface FLT3 and were therefore not enriched to a statistically significant extent with the bright versus negative sorting scheme. Thus, clear segregation of the most primitive progenitors from BM CD34+ cells was confounded by low apparent levels of FLT3 cell-surface expression on these cells, whereas myeloid progenitors unambiguously segregated with the FLT3 brightest cells and erythroid progenitors with the FLT3 dimmest. Additional phenotypic analyses using MoAbs against progenitor/stem cell markers including the mucinlike molecule MGC-24v (CD164), the receptor tyrosine kinases TIE, FMS (CD115), and KIT (CD117) further illustrate the differences in surface antigen expression profiles of BM and CB CD34+cells. Notably, CD115 is rarely detected on CB CD34+cells, whereas 20% to 25% of the BM CD34+FLT3+ cells are CD115+. Furthermore, 80% to 95% of the CB CD34+CD117+ but only 60% to 75% of the BM CD34+CD117+ cells coexpress FLT3. Only a negligible amount of CD34+CD19+ are detected in CB, while in BM 20% to 30% of CD34+CD19+ presumed pro/pre-B cells coexpress FLT3. In contrast, the majority of the CD34+CD164+ and CD34+TIE+ subsets in both CB and BM coexpress FLT3. Analysis of unseparated cells showed that FLT3 expression is not restricted to CD34+ subsets. About 65% to 70% of lymphocyte-gated BM CD34−FLT3+ cells are positive for the monocytic marker CD115 whereas 25% to 30% of these cells consist of CD10 expressing B-cell precursors. Finally, CD34−monocytes in BM, CB, and PB express FLT3 whereas granulocytes are FLT3−. Our data show that detectable FLT3 appears first at low levels on the surface of primitive multilineage progenitor cells and disappears during defined stages of B-cell development, but is upregulated and maintained during monocytic maturation. © 1997 by The American Society of Hematology.

1009 – HSPC EDITING IN THE RHESUS MACAQUE TO MODEL HUMAN BIOLOGY AND TO DEVELOP NEW THERAPIES

The rhesus macaque autologous transplantation model has been predictive and instructive in better understanding human hematopoiesis and translating novel gene and cell therapies. We have developed a robust CRISPR/Cas9 gene editing platform in this model for a variety of applications. Engraftment of multilineage hematopoietic stem and progenitor cells with up to 90% of edited knockout alleles has been achieved in macaques. We have created a macaque model of age-related clonal hematopoiesis(ARCH) following autologous transplantation of edited HSPC in macaques, demonstrating reproducible clonal expansion of TET2 mutant cells, and gained insights into hematopoietic and cardiovascular manifestations of ARCH, along with using the model to investigate potential therapeutic interventions able to inhibit clonal expansion. We have pursued knockout of CD33 as an approach to protect normal HSPC from CAR-T attack, providing a pathway to safe and effective utilization of CAR-T to target myeloid malignancies. Finally, we have compared approaches to predict off-target effects in normal primary engrafting HSPC, investigating whether <i>in silico</i> algorithm prediction or CircleSeq, a method for selective sequencing of DNA exposed to nucleases <i>in vitro</i> is better able to identify valid off-target sites found in vivo in macaques following autologous transplantation.

Development of immortalized human hepatocyte-like hybrid cells by fusion of multi-lineage progenitor cells with primary hepatocytes.

Human primary hepatocytes (PHs) are critical to studying liver functions, drug metabolism and toxicity. PHs isolated from livers that are unacceptable for transplantation have limited expansion and culture viability in vitro, in addition to rapidly deteriorating enzymatic functions. The unsuitability of immortalized hepato-carcinoma cell lines for this function has prompted studies to develop hepatocyte-like cells from alternative sources like ESC, iPS, and other stem cell types using differentiation protocols. This study describes a novel technique to produce expandable and functional hepatocyte-like cells from the fusion of an immortalized human umbilical cord blood derived cell line (E12 MLPC) to normal human primary hepatocytes. Multi-lineage progenitor cells (MLPC) comprise a small subset of mesenchymal-like cells isolated from human umbilical cord blood. MLPC are distinguishable from other mesenchymal-like cells by their extended expansion capacity (up to 80 cell doublings before senescence) and the ability to be differentiated into cells representative of endo-, meso- and ectodermal origins. Transfection of MLPC with the gene for telomerase reverse transcriptase (TERT) resulted in clonal cell lines that were capable of differentiation to different cellular outcomes while maintaining their functional immortality. A methodology for the development of immortalized hepatocyte-like hybrid cells by the in vitro fusion of human MLPC with normal human primary hepatocytes is reported. The resultant hybrid cells exhibited homology with hepatocytes by morphology, immunohistochemistry, urea and albumin production and gene expression. A medium that allows stable long-term expansion of hepatocyte-like fusion cells is described.

In vitro Differentiation of Human TERT-Transfected Multi-Lineage Progenitor Cells (MLPC) into Immortalized Hepatocyte-Like Cells.

BackgroundResearch directed towards drug development, metabolism, and liver functions often utilize primary hepatocytes (PH) for preliminary in vitro studies. Variability in the in vitro functionality of PH and the unsuitability of hepatocarcinoma cells for these studies have driven researchers to look to ESC, iPS, and other stem cell types using differentiation protocols to provide more reliable and available cells. This study describes the development of hepatocyte-like cells through the in vitro differentiation of human TERT-immortalized cord blood-derived multi-lineage progenitor cells (MLPC). The E12 clonal cell line derived from polyclonal TERT-transfected cells was used throughout the study.MethodsE12 MLPC were subjected to a three-step differentiation protocol using alternating combinations of growth factors, cytokines, and maturational factors. Cells at various stages of differentiation were analyzed for consistency with PH by morphology, immunohistochemistry, urea production, and gene expression.ResultsE12 MLPC were shown to significantly change morphology with each stage of differentiation. Coincidental with the morphological changes in the cells, immunohistochemistry data documented the differentiation to committed endoderm by the expression of SOX-17 and GATA-4; the progression to committed hepatocyte-like cells by the expression of a large number of markers including α-fetoprotein and albumin; and the final differentiation by the expression of nuclear and cytoplasmic HNF4. Fully differentiated cells demonstrated gene expression, urea production, and immunohistochemistry consistent with PH. A methodology and medium formulation to continuously expand the E12-derived hepatocyte-like cells is described.ConclusionThe availability of immortalized hepatocyte-like cell lines could provide a consistent tool for the study of hepatic diseases, drug discovery, and the development of cellular therapies for liver disorders. Utilization of these techniques could provide a basis for the development of bridge therapies for liver failure patients awaiting transplant.

Pericytes in Microvessels: From "Mural" Function to Brain and Retina Regeneration.

Pericytes are branched cells located in the wall of capillary blood vessels that are found throughout the body, embedded within the microvascular basement membrane and wrapping endothelial cells, with which they establish a strong physical contact. Pericytes regulate angiogenesis, vessel stabilization, and contribute to the formation of both the blood-brain and blood-retina barriers by Angiopoietin-1/Tie-2, platelet derived growth factor (PDGF) and transforming growth factor (TGF) signaling pathways, regulating pericyte-endothelial cell communication. Human pericytes that have been cultured for a long period give rise to multilineage progenitor cells and exhibit mesenchymal stem cell (MSC) features. We focused our attention on the roles of pericytes in brain and ocular diseases. In particular, pericyte involvement in brain ischemia, brain tumors, diabetic retinopathy, and uveal melanoma is described. Several molecules, such as adenosine and nitric oxide, are responsible for pericyte shrinkage during ischemia-reperfusion. Anti-inflammatory molecules, such as IL-10, TGFβ, and MHC-II, which are increased in glioblastoma-activated pericytes, are responsible for tumor growth. As regards the eye, pericytes play a role not only in ocular vessel stabilization, but also as a stem cell niche that contributes to regenerative processes in diabetic retinopathy. Moreover, pericytes participate in melanoma cell extravasation and the genetic ablation of the PDGF receptor reduces the number of pericytes and aberrant tumor microvessel formation with important implications for therapy efficacy. Thanks to their MSC features, pericytes could be considered excellent candidates to promote nervous tissue repair and for regenerative medicine.

Systematic integration of GATA transcription factors and epigenomes via IDEAS paints the regulatory landscape of hematopoietic cells.

Members of the GATA family of transcription factors play key roles in the differentiation of specific cell lineages by regulating the expression of target genes. Three GATA factors play distinct roles in hematopoietic differentiation. In order to better understand how these GATA factors function to regulate genes throughout the genome, we are studying the epigenomic and transcriptional landscapes of hematopoietic cells in a model‐driven, integrative fashion. We have formed the collaborative multi‐lab VISION project to conduct ValIdated Systematic IntegratiON of epigenomic data in mouse and human hematopoiesis. The epigenomic data included nuclease accessibility in chromatin, CTCF occupancy, and histone H3 modifications for 20 cell types covering hematopoietic stem cells, multilineage progenitor cells, and mature cells across the blood cell lineages of mouse. The analysis used the Integrative and Discriminative Epigenome Annotation System (IDEAS), which learns all common combinations of features (epigenetic states) simultaneously in two dimensions—along chromosomes and across cell types. The result is a segmentation that effectively paints the regulatory landscape in readily interpretable views, revealing constitutively active or silent loci as well as the loci specifically induced or repressed in each stage and lineage. Nuclease accessible DNA segments in active chromatin states were designated candidate cis‐regulatory elements in each cell type, providing one of the most comprehensive registries of candidate hematopoietic regulatory elements to date. Applications of VISION resources are illustrated for the regulation of genes encoding GATA1, GATA2, GATA3, and Ikaros. VISION resources are freely available from our website http://usevision.org.

Periodontal Regeneration by Allogeneic Transplantation of Adipose Tissue Derived Multi-Lineage Progenitor Stem Cells in vivo

The ultimate goal of periodontal disease treatment is the reorganization of functional tissue that can regenerate lost periodontal tissue. Regeneration of periodontal tissues is clinically possible by using autogenic transplantation of MSCs. However, autologous MSC transplantation is limited depending on age, systemic disease and tissue quality, thus precluding their clinical application. Therefore, we evaluated the efficacy of allogeneic transplantation of adipose-derived multi-lineage progenitor cells (ADMPC) in a micro-mini pig periodontal defect model. ADMPC were isolated from the greater omentum of micro-mini pigs, and flow cytometry analysis confirmed that the ADMPC expressed MSC markers, including CD44 and CD73. ADMPC exhibited osteogenic, adipogenic and periodontal ligament differentiation capacities in differentiation medium. ADMPC showed high expression of the immune suppressive factors GBP4 and IL1-RA upon treatment with a cytokine cocktail containing interferon-γ, tumor necrosis factor-α and interleukin-6. Allogeneic transplantation of ADMPC in a micro-mini pig periodontal defect model showed significant bone regeneration ability based on bone-morphometric analysis. Moreover, the regeneration ability of ADMPC by allogeneic transplantation was comparable to those of autologous transplantation by histological analysis. These results indicate that ADMPC have immune-modulation capability that can induce periodontal tissue regeneration by allogeneic transplantation.

In Vivo Rescue Assay of RhoA-Deficient Hematopoietic Stem and Progenitor Cells.

RhoA is a founding member of Rho GTPase family and is a well-recognized regulator of actin cytoskeleton dynamics. Signal pathways transduced by RhoA are involved in cell migration, polarity, morphogenesis, proliferation, survival, and cell fate decision. Conditional gene targeting of Rhoa in murine blood system induces acute hematopoietic failure due to the loss of multilineage hematopoietic progenitor cells (HPC) caused by a cytokinesis defect and necrosis. Here we describe a method to conditionally induce Rhoa gene knockout in murine blood cells and a rescue by exogenous RhoA expression with lentivirus in HPCs, an approach that has general applicability in studying in vivo function of Rho GTPases and their regulators/effectors by gene targeting.