Publications by authors named "Hanna K A Mikkola"

51 Publications

Nidogen-1 Mitigates Ischemia and Promotes Tissue Survival and Regeneration.

Adv Sci (Weinh) 2021 Feb 21;8(4):2002500. Epub 2020 Dec 21.

Department of Bioengineering Eberhard Karls University Tübingen Tübingen 72076 Germany.

Ischemia impacts multiple organ systems and is the major cause of morbidity and mortality in the developed world. Ischemia disrupts tissue homeostasis, driving cell death, and damages tissue structure integrity. Strategies to heal organs, like the infarcted heart, or to replace cells, as done in pancreatic islet -cell transplantations, are often hindered by ischemic conditions. Here, it is discovered that the basement membrane glycoprotein nidogen-1 attenuates the apoptotic effect of hypoxia in cardiomyocytes and pancreatic -cells via the v3 integrin and beneficially modulates immune responses in vitro. It is shown that nidogen-1 significantly increases heart function and angiogenesis, while reducing fibrosis, in a mouse postmyocardial infarction model. These results demonstrate the protective and regenerative potential of nidogen-1 in ischemic conditions.
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http://dx.doi.org/10.1002/advs.202002500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7887579PMC
February 2021

MLLT3 governs human haematopoietic stem-cell self-renewal and engraftment.

Nature 2019 12 27;576(7786):281-286. Epub 2019 Nov 27.

Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, CA, USA.

Limited knowledge of the mechanisms that govern the self-renewal of human haematopoietic stem cells (HSCs), and why this fails in culture, have impeded the expansion of HSCs for transplantation. Here we identify MLLT3 (also known as AF9) as a crucial regulator of HSCs that is highly enriched in human fetal, neonatal and adult HSCs, but downregulated in culture. Depletion of MLLT3 prevented the maintenance of transplantable human haematopoietic stem or progenitor cells (HSPCs) in culture, whereas stabilizing MLLT3 expression in culture enabled more than 12-fold expansion of transplantable HSCs that provided balanced multilineage reconstitution in primary and secondary mouse recipients. Similar to endogenous MLLT3, overexpressed MLLT3 localized to active promoters in HSPCs, sustained levels of H3K79me2 and protected the HSC transcriptional program in culture. MLLT3 thus acts as HSC maintenance factor that links histone reader and modifying activities to modulate HSC gene expression, and may provide a promising approach to expand HSCs for transplantation.
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http://dx.doi.org/10.1038/s41586-019-1790-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278275PMC
December 2019

Protagonist or antagonist? The complex roles of retinoids in the regulation of hematopoietic stem cells and their specification from pluripotent stem cells.

Exp Hematol 2018 09 4;65:1-16. Epub 2018 Jul 4.

St. Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia; The University of Melbourne, Department of Medicine at St. Vincent's Hospital, Fitzroy, Victoria, Australia. Electronic address:

Hematopoietic stem cells (HSCs) are multipotent cells responsible for the maintenance of the hematopoietic system throughout life. Dysregulation of the balance in HSC self-renewal, death, and differentiation can have serious consequences such as myelodysplastic syndromes or leukemia. All-trans retinoic acid (ATRA), the biologically active metabolite of vitamin A/RA, has been shown to have pleiotropic effects on hematopoietic cells, enhancing HSC self-renewal while also increasing differentiation of more mature progenitors. Furthermore, ATRA has been shown to have key roles in regulating the specification and formation of hematopoietic cells from pluripotent stem cells including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Here, we summarize the known roles of vitamin A and RA receptors in the regulation of hematopoiesis from HSCs, ES, and iPSCs.
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http://dx.doi.org/10.1016/j.exphem.2018.06.287DOI Listing
September 2018

Genetic Regulation of Fibroblast Activation and Proliferation in Cardiac Fibrosis.

Circulation 2018 09;138(12):1224-1235

Division of Cardiology, Department of Medicine, David Geffen School of Medicine (S.P., S.R., P.Z., M.J.M., J.S.D., A.H.-V., X.W., R.Q., J.M.S., A.J.L., R.A.), University of California, Los Angeles.

Background: Genetic diversity and the heterogeneous nature of cardiac fibroblasts (CFbs) have hindered characterization of the molecular mechanisms that regulate cardiac fibrosis. The Hybrid Mouse Diversity Panel offers a valuable tool to examine genetically diverse cardiac fibroblasts and their role in fibrosis.

Methods: Three strains of mice (C57BL/6J, C3H/HeJ, and KK/HlJ) were selected from the Hybrid Mouse Diversity Panel and treated with either isoproterenol (ISO) or saline by an intraperitoneally implanted osmotic pump. After 21 days, cardiac function and levels of fibrosis were measured by echocardiography and trichrome staining, respectively. Activation and proliferation of CFbs were measured by in vitro and in vivo assays under normal and injury conditions. RNA sequencing was done on isolated CFbs from each strain. Results were analyzed by Ingenuity Pathway Analysis and validated by reverse transcription-qPCR, immunohistochemistry, and ELISA.

Results: ISO treatment in C57BL/6J, C3H/HeJ, and KK/HlJ mice resulted in minimal, moderate, and extensive levels of fibrosis, respectively (n=7-8 hearts per condition). Isolated CFbs treated with ISO exhibited strain-specific increases in the levels of activation but showed comparable levels of proliferation. Similar results were found in vivo, with fibroblast activation, and not proliferation, correlating with the differential levels of cardiac fibrosis after ISO treatment. RNA sequencing revealed that CFbs from each strain exhibit unique gene expression changes in response to ISO. We identified Ltbp2 as a commonly upregulated gene after ISO treatment. Expression of LTBP2 was elevated and specifically localized in the fibrotic regions of the myocardium after injury in mice and in human heart failure patients.

Conclusions: This study highlights the importance of genetic variation in cardiac fibrosis by using multiple inbred mouse strains to characterize CFbs and their response to ISO treatment. Our data suggest that, although fibroblast activation is a response that parallels the extent of scar formation, proliferation may not necessarily correlate with levels of fibrosis. In addition, by comparing CFbs from multiple strains, we identified pathways as potential therapeutic targets and LTBP2 as a marker for fibrosis, with relevance to patients with underlying myocardial fibrosis.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.118.035420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6202226PMC
September 2018

Analysis of cardiomyocyte clonal expansion during mouse heart development and injury.

Nat Commun 2018 02 21;9(1):754. Epub 2018 Feb 21.

Division of Cardiology, Department of Internal Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, 90095, USA.

The cellular mechanisms driving cardiac tissue formation remain poorly understood, largely due to the structural and functional complexity of the heart. It is unclear whether newly generated myocytes originate from cardiac stem/progenitor cells or from pre-existing cardiomyocytes that re-enter the cell cycle. Here, we identify the source of new cardiomyocytes during mouse development and after injury. Our findings suggest that cardiac progenitors maintain proliferative potential and are the main source of cardiomyocytes during development; however, the onset of αMHC expression leads to reduced cycling capacity. Single-cell RNA sequencing reveals a proliferative, "progenitor-like" population abundant in early embryonic stages that decreases to minimal levels postnatally. Furthermore, cardiac injury by ligation of the left anterior descending artery was found to activate cardiomyocyte proliferation in neonatal but not adult mice. Our data suggest that clonal dominance of differentiating progenitors mediates cardiac development, while a distinct subpopulation of cardiomyocytes may have the potential for limited proliferation during late embryonic development and shortly after birth.
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http://dx.doi.org/10.1038/s41467-018-02891-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5821855PMC
February 2018

Hepatic Leukemia Factor Maintains Quiescence of Hematopoietic Stem Cells and Protects the Stem Cell Pool during Regeneration.

Cell Rep 2017 Dec;21(12):3514-3523

Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, BMC A12, 221 84, Lund, Sweden. Electronic address:

The transcription factor hepatic leukemia factor (HLF) is strongly expressed in hematopoietic stem cells (HSCs) and is thought to influence both HSC self-renewal and leukemogenesis. However, the physiological role of HLF in hematopoiesis and HSC function is unclear. Here, we report that mice lacking Hlf are viable with essentially normal hematopoietic parameters, including an intact HSC pool during steady-state hematopoiesis. In contrast, when challenged through transplantation, Hlf-deficient HSCs showed an impaired ability to reconstitute hematopoiesis and became gradually exhausted upon serial transplantation. Transcriptional profiling of Hlf-deficient HSCs revealed changes associated with enhanced cellular activation, and cell-cycle analysis demonstrated a significant reduction of quiescent HSCs. Accordingly, toxic insults targeting dividing cells completely eradicated the HSC pool in Hlf-deficient mice. In summary, our findings point to HLF as a critical regulator of HSC quiescence and as an essential factor for maintaining the HSC pool during regeneration.
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http://dx.doi.org/10.1016/j.celrep.2017.11.084DOI Listing
December 2017

LYVE1 Marks the Divergence of Yolk Sac Definitive Hemogenic Endothelium from the Primitive Erythroid Lineage.

Cell Rep 2016 11;17(9):2286-2298

Department of Molecular, Cell & Developmental Biology, UCLA, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, UCLA, Los Angeles, CA 90095, USA. Electronic address:

The contribution of the different waves and sites of developmental hematopoiesis to fetal and adult blood production remains unclear. Here, we identify lymphatic vessel endothelial hyaluronan receptor-1 (LYVE1) as a marker of yolk sac (YS) endothelium and definitive hematopoietic stem and progenitor cells (HSPCs). Endothelium in mid-gestation YS and vitelline vessels, but not the dorsal aorta and placenta, were labeled by Lyve1-Cre. Most YS HSPCs and erythro-myeloid progenitors were Lyve1-Cre lineage traced, but primitive erythroid cells were not, suggesting that they represent distinct lineages. Fetal liver (FL) and adult HSPCs showed 35%-40% Lyve1-Cre marking. Analysis of circulation-deficient Ncx1 concepti identified the YS as a major source of Lyve1-Cre labeled HSPCs. FL proerythroblast marking was extensive at embryonic day (E) 11.5-13.5, but decreased to hematopoietic stem cell (HSC) levels by E16.5, suggesting that HSCs from multiple sources became responsible for erythropoiesis. Lyve1-Cre thus marks the divergence between YS primitive and definitive hematopoiesis and provides a tool for targeting YS definitive hematopoiesis and FL colonization.
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http://dx.doi.org/10.1016/j.celrep.2016.10.080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6940422PMC
November 2016

Differentiation of human embryonic stem cells to HOXA hemogenic vasculature that resembles the aorta-gonad-mesonephros.

Nat Biotechnol 2016 Nov 17;34(11):1168-1179. Epub 2016 Oct 17.

Murdoch Childrens Research Institute, The Royal Children's Hospital, Parkville, Victoria, Australia.

The ability to generate hematopoietic stem cells from human pluripotent cells would enable many biomedical applications. We find that hematopoietic CD34 cells in spin embryoid bodies derived from human embryonic stem cells (hESCs) lack HOXA expression compared with repopulation-competent human cord blood CD34 cells, indicating incorrect mesoderm patterning. Using reporter hESC lines to track the endothelial (SOX17) to hematopoietic (RUNX1C) transition that occurs in development, we show that simultaneous modulation of WNT and ACTIVIN signaling yields CD34 hematopoietic cells with HOXA expression that more closely resembles that of cord blood. The cultures generate a network of aorta-like SOX17 vessels from which RUNX1C blood cells emerge, similar to hematopoiesis in the aorta-gonad-mesonephros (AGM). Nascent CD34 hematopoietic cells and corresponding cells sorted from human AGM show similar expression of cell surface receptors, signaling molecules and transcription factors. Our findings provide an approach to mimic in vitro a key early stage in human hematopoiesis for the generation of AGM-derived hematopoietic lineages from hESCs.
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http://dx.doi.org/10.1038/nbt.3702DOI Listing
November 2016

MEF2C protects bone marrow B-lymphoid progenitors during stress haematopoiesis.

Nat Commun 2016 08 10;7:12376. Epub 2016 Aug 10.

Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California 90095, USA.

DNA double strand break (DSB) repair is critical for generation of B-cell receptors, which are pre-requisite for B-cell progenitor survival. However, the transcription factors that promote DSB repair in B cells are not known. Here we show that MEF2C enhances the expression of DNA repair and recombination factors in B-cell progenitors, promoting DSB repair, V(D)J recombination and cell survival. Although Mef2c-deficient mice maintain relatively intact peripheral B-lymphoid cellularity during homeostasis, they exhibit poor B-lymphoid recovery after sub-lethal irradiation and 5-fluorouracil injection. MEF2C binds active regulatory regions with high-chromatin accessibility in DNA repair and V(D)J genes in both mouse B-cell progenitors and human B lymphoblasts. Loss of Mef2c in pre-B cells reduces chromatin accessibility in multiple regulatory regions of the MEF2C-activated genes. MEF2C therefore protects B lymphopoiesis during stress by ensuring proper expression of genes that encode DNA repair and B-cell factors.
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http://dx.doi.org/10.1038/ncomms12376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4987520PMC
August 2016

Critical requirement of VEGF-C in transition to fetal erythropoiesis.

Blood 2016 08 24;128(5):710-20. Epub 2016 Jun 24.

Wihuri Research Institute and Translational Cancer Biology Program, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland; and.

Vascular endothelial growth factor C (VEGF-C) is a major driver of lymphangiogenesis in embryos and adults. Vegfc gene deletion in mouse embryos results in failure of lymphangiogenesis, fluid accumulation in tissues, and lethality. The VEGF-C receptors VEGFR3 and VEGFR2 are required for embryonic blood vessel formation. The related VEGF is essential for both blood vessel formation and embryonic hematopoiesis, whereas the possible involvement of VEGF-C in hematopoiesis is unknown. Here we unveil a novel hematopoietic function of VEGF-C in fetal erythropoiesis. Deletion of Vegfc in embryonic day 7.5 (E7.5) embryos in the C57BL6 mouse genetic background led to defective fetal erythropoiesis, characterized by anemia and lack of enucleated red blood cells in blood circulation. Macrophages and erythroid cells in the fetal liver (FL) were also decreased after midgestation because of decreased cell proliferation and increased apoptosis. However, the Lin(-)Sca-1(+)c-Kit(+) stem cell compartment in E14.5 FL was not affected by Vegfc deletion. VEGF-C loss did not disrupt the generation of primitive erythroid cells or erythro-myeloid progenitors (EMPs) in the yolk sac, but it decreased the expression of α4-integrin on EMPs and compromised EMP colonization of the FL. The distribution, maturation, and enucleation of primitive erythroblasts were also impaired by Vegfc deletion. In contrast, Vegfc deletion from E10.5 onward did not compromise definitive hematopoiesis in the liver, and Vegfc deletion in adult mice did not cause anemia. These results reveal an unexpected role for VEGF-C, a major lymphangiogenic growth factor, in the transition to FL erythropoiesis.
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http://dx.doi.org/10.1182/blood-2015-12-687970DOI Listing
August 2016

Medial HOXA genes demarcate haematopoietic stem cell fate during human development.

Nat Cell Biol 2016 06 16;18(6):595-606. Epub 2016 May 16.

Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, California 90095, USA.

Pluripotent stem cells (PSCs) may provide a potential source of haematopoietic stem/progenitor cells (HSPCs) for transplantation; however, unknown molecular barriers prevent the self-renewal of PSC-HSPCs. Using two-step differentiation, human embryonic stem cells (hESCs) differentiated in vitro into multipotent haematopoietic cells that had the CD34(+)CD38(-/lo)CD90(+)CD45(+)GPI-80(+) fetal liver (FL) HSPC immunophenotype, but exhibited poor expansion potential and engraftment ability. Transcriptome analysis of immunophenotypic hESC-HSPCs revealed that, despite their molecular resemblance to FL-HSPCs, medial HOXA genes remained suppressed. Knockdown of HOXA7 disrupted FL-HSPC function and caused transcriptome dysregulation that resembled hESC-derived progenitors. Overexpression of medial HOXA genes prolonged FL-HSPC maintenance but was insufficient to confer self-renewal to hESC-HSPCs. Stimulation of retinoic acid signalling during endothelial-to-haematopoietic transition induced the HOXA cluster and other HSC/definitive haemogenic endothelium genes, and prolonged HSPC maintenance in culture. Thus, medial HOXA gene expression induced by retinoic acid signalling marks the establishment of the definitive HSPC fate and controls HSPC identity and function.
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http://dx.doi.org/10.1038/ncb3354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4981340PMC
June 2016

Tracking HSC Origin: From Bench to Placenta.

Dev Cell 2016 Mar;36(5):479-80

Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of California Los Angeles, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA 90095, USA. Electronic address:

Reporting in Developmental Cell, Pereira et al. (2016) use in vitro lineage reprogramming insights to inform understanding of hematopoietic stem cell (HSC) development in vivo. They find Prom1(+)Sca1(+)CD34(+)CD45(-) hemogenic precursors, akin to fibroblast-derived hemato-vascular precursors, in mouse placenta and embryo. The cells mature into transplantable HSCs in culture.
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http://dx.doi.org/10.1016/j.devcel.2016.02.022DOI Listing
March 2016

GLI2 inhibition abrogates human leukemia stem cell dormancy.

J Transl Med 2015 Mar 21;13:98. Epub 2015 Mar 21.

Department of Medicine, Stem Cell Program and Moores Cancer Center, University of California San Diego, 3855 Health Sciences Drive, La Jolla, 92037, CA, USA.

Background: Dormant leukemia stem cells (LSC) promote therapeutic resistance and leukemic progression as a result of unbridled activation of stem cell gene expression programs. Thus, we hypothesized that 1) deregulation of the hedgehog (Hh) stem cell self-renewal and cell cycle regulatory pathway would promote dormant human LSC generation and 2) that PF-04449913, a clinical antagonist of the GLI2 transcriptional activator, smoothened (SMO), would enhance dormant human LSC eradication.

Methods: To test these postulates, whole transcriptome RNA sequencing (RNA-seq), microarray, qRT-PCR, stromal co-culture, confocal fluorescence microscopic, nanoproteomic, serial transplantation and cell cycle analyses were performed on FACS purified normal, chronic phase (CP) chronic myeloid leukemia (CML), blast crisis (BC) phase CML progenitors with or without PF-04449913 treatment.

Results: Notably, RNA-seq analyses revealed that Hh pathway and cell cycle regulatory gene overexpression correlated with leukemic progression. While lentivirally enforced GLI2 expression enhanced leukemic progenitor dormancy in stromal co-cultures, this was not observed with a mutant GLI2 lacking a transactivation domain, suggesting that GLI2 expression prevented cell cycle transit. Selective SMO inhibition with PF-04449913 in humanized stromal co-cultures and LSC xenografts reduced downstream GLI2 protein and cell cycle regulatory gene expression. Moreover, SMO inhibition enhanced cell cycle transit and sensitized BC LSC to tyrosine kinase inhibition in vivo at doses that spare normal HSC.

Conclusion: In summary, while GLI2, forms part of a core HH pathway transcriptional regulatory network that promotes human myeloid leukemic progression and dormant LSC generation, selective inhibition with PF-04449913 reduces the dormant LSC burden thereby providing a strong rationale for clinical trials predicated on SMO inhibition in combination with TKIs or chemotherapeutic agents with the ultimate aim of obviating leukemic therapeutic resistance, persistence and progression.
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http://dx.doi.org/10.1186/s12967-015-0453-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4414375PMC
March 2015

The histone methyltransferase activity of MLL1 is dispensable for hematopoiesis and leukemogenesis.

Cell Rep 2014 May 9;7(4):1239-47. Epub 2014 May 9.

Department of Genetics, Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA; Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA; Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA. Electronic address:

Despite correlations between histone methyltransferase (HMT) activity and gene regulation, direct evidence that HMT activity is responsible for gene activation is sparse. We address the role of the HMT activity for MLL1, a histone H3 lysine 4 (H3K4) methyltransferase critical for maintaining hematopoietic stem cells (HSCs). Here, we show that the SET domain, and thus HMT activity of MLL1, is dispensable for maintaining HSCs and supporting leukemogenesis driven by the MLL-AF9 fusion oncoprotein. Upon Mll1 deletion, histone H4 lysine 16 (H4K16) acetylation is selectively depleted at MLL1 target genes in conjunction with reduced transcription. Surprisingly, inhibition of SIRT1 is sufficient to prevent the loss of H4K16 acetylation and the reduction in MLL1 target gene expression. Thus, recruited MOF activity, and not the intrinsic HMT activity of MLL1, is central for the maintenance of HSC target genes. In addition, this work reveals a role for SIRT1 in opposing MLL1 function.
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http://dx.doi.org/10.1016/j.celrep.2014.04.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4120120PMC
May 2014

Sex hormone drives blood stem cell reproduction.

EMBO J 2014 Mar 20;33(6):534-5. Epub 2014 Feb 20.

Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, CA, USA.

Stem cells ensure the maintenance of tissue homeostasis throughout life by tightly regulating their self-renewal and differentiation. In a recent study published in Nature, Nakada et al, 2014 unveil an unexpected endocrine mechanism that regulates hematopoietic stem cell (HSC) self-renewal.
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http://dx.doi.org/10.1002/embj.201487976DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3989647PMC
March 2014

Progesterone receptor in the vascular endothelium triggers physiological uterine permeability preimplantation.

Cell 2014 Jan;156(3):549-62

Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address:

Vascular permeability is frequently associated with inflammation and is triggered by a cohort of secreted permeability factors such as vascular endothelial growth factor (VEGF). Here, we show that the physiological vascular permeability that precedes implantation is directly controlled by progesterone receptor (PR) and is independent of VEGF. Global or endothelial-specific deletion of PR blocks physiological vascular permeability in the uterus, whereas misexpression of PR in the endothelium of other organs results in ectopic vascular leakage. Integration of an endothelial genome-wide transcriptional profile with chromatin immunoprecipitation sequencing revealed that PR induces an NR4A1 (Nur77/TR3)-dependent transcriptional program that broadly regulates vascular permeability in response to progesterone. Silencing of NR4A1 blocks PR-mediated permeability responses, indicating a direct link between PR and NR4A1. This program triggers concurrent suppression of several junctional proteins and leads to an effective, timely, and venous-specific regulation of vascular barrier function that is critical for embryo implantation.
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http://dx.doi.org/10.1016/j.cell.2013.12.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985399PMC
January 2014

c-Met-dependent multipotent labyrinth trophoblast progenitors establish placental exchange interface.

Dev Cell 2013 Nov;27(4):373-86

Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA.

The placenta provides the interface for gas and nutrient exchange between the mother and the fetus. Despite its critical function in sustaining pregnancy, the stem/progenitor cell hierarchy and molecular mechanisms responsible for the development of the placental exchange interface are poorly understood. We identified an Epcam(hi) labyrinth trophoblast progenitor (LaTP) in mouse placenta that at a clonal level generates all labyrinth trophoblast subtypes, syncytiotrophoblasts I and II, and sinusoidal trophoblast giant cells. Moreover, we discovered that hepatocyte growth factor/c-Met signaling is required for sustaining proliferation of LaTP during midgestation. Loss of trophoblast c-Met also disrupted terminal differentiation and polarization of syncytiotrophoblasts, leading to intrauterine fetal growth restriction, fetal liver hypocellularity, and demise. Identification of this c-Met-dependent multipotent LaTP provides a landmark in the poorly defined placental stem/progenitor cell hierarchy and may help us understand pregnancy complications caused by a defective placental exchange.
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http://dx.doi.org/10.1016/j.devcel.2013.10.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3950757PMC
November 2013

Selective suppression of endothelial cytokine production by progesterone receptor.

Vascul Pharmacol 2013 Jul-Aug;59(1-2):36-43. Epub 2013 Jun 6.

Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA.

Steroid hormones are well-recognized suppressors of the inflammatory response, however, their cell- and tissue-specific effects in the regulation of inflammation are far less understood, particularly for the sex-related steroids. To determine the contribution of progesterone in the endothelium, we have characterized and validated an in vitro culture system in which human umbilical vein endothelial cells constitutively express human progesterone receptor (PR). Using next generation RNA-sequencing, we identified a selective group of cytokines that are suppressed by progesterone both under physiological conditions and during pathological activation by lipopolysaccharide. In particular, IL-6, IL-8, CXCL2/3, and CXCL1 were found to be direct targets of PR, as determined by ChIP-sequencing. Regulation of these cytokines by progesterone was also confirmed by bead-based multiplex cytokine assays and quantitative PCR. These findings provide a novel role for PR in the direct regulation of cytokine levels secreted by the endothelium. They also suggest that progesterone-PR signaling in the endothelium directly impacts leukocyte trafficking in PR-expressing tissues.
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http://dx.doi.org/10.1016/j.vph.2013.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3819224PMC
March 2014

Haemogenic endocardium contributes to transient definitive haematopoiesis.

Nat Commun 2013 ;4:1564

Department of Molecular Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, California 90095, USA.

Haematopoietic cells arise from spatiotemporally restricted domains in the developing embryo. Although studies of non-mammalian animal and in vitro embryonic stem cell models suggest a close relationship among cardiac, endocardial and haematopoietic lineages, it remains unknown whether the mammalian heart tube serves as a haemogenic organ akin to the dorsal aorta. Here we examine the haemogenic activity of the developing endocardium. Mouse heart explants generate myeloid and erythroid colonies in the absence of circulation. Haemogenic activity arises from a subset of endocardial cells in the outflow cushion and atria earlier than in the aorta-gonad-mesonephros region, and is transient and definitive in nature. Interestingly, key cardiac transcription factors, Nkx2-5 and Isl1, are expressed in and required for the haemogenic population of the endocardium. Together, these data suggest that a subset of endocardial/endothelial cells serve as a de novo source for transient definitive haematopoietic progenitors.
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http://dx.doi.org/10.1038/ncomms2569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3612528PMC
June 2013

Expansion on stromal cells preserves the undifferentiated state of human hematopoietic stem cells despite compromised reconstitution ability.

PLoS One 2013 16;8(1):e53912. Epub 2013 Jan 16.

Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of America.

Lack of HLA-matched hematopoietic stem cells (HSC) limits the number of patients with life-threatening blood disorders that can be treated by HSC transplantation. So far, insufficient understanding of the regulatory mechanisms governing human HSC has precluded the development of effective protocols for culturing HSC for therapeutic use and molecular studies. We defined a culture system using OP9M2 mesenchymal stem cell (MSC) stroma that protects human hematopoietic stem/progenitor cells (HSPC) from differentiation and apoptosis. In addition, it facilitates a dramatic expansion of multipotent progenitors that retain the immunophenotype (CD34+CD38-CD90+) characteristic of human HSPC and proliferative potential over several weeks in culture. In contrast, transplantable HSC could be maintained, but not significantly expanded, during 2-week culture. Temporal analysis of the transcriptome of the ex vivo expanded CD34+CD38-CD90+ cells documented remarkable stability of most transcriptional regulators known to govern the undifferentiated HSC state. Nevertheless, it revealed dynamic fluctuations in transcriptional programs that associate with HSC behavior and may compromise HSC function, such as dysregulation of PBX1 regulated genetic networks. This culture system serves now as a platform for modeling human multilineage hematopoietic stem/progenitor cell hierarchy and studying the complex regulation of HSC identity and function required for successful ex vivo expansion of transplantable HSC.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0053912PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3547050PMC
July 2013

Nipah virus envelope-pseudotyped lentiviruses efficiently target ephrinB2-positive stem cell populations in vitro and bypass the liver sink when administered in vivo.

J Virol 2013 Feb 28;87(4):2094-108. Epub 2012 Nov 28.

Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, California, USA.

Sophisticated retargeting systems for lentiviral vectors have been developed in recent years. Most seek to suppress the viral envelope's natural tropism while modifying the receptor-binding domain such that its tropism is determined by the specificity of the engineered ligand-binding motif. Here we took advantage of the natural tropism of Nipah virus (NiV), whose attachment envelope glycoprotein has picomolar affinity for ephrinB2, a molecule proposed as a molecular marker of "stemness" (present on embryonic, hematopoietic, and neural stem cells) as well as being implicated in tumorigenesis of specific cancers. NiV entry requires both the fusion (F) and attachment (G) glycoproteins. Truncation of the NiV-F cytoplasmic tail (T5F) alone, combined with full-length NiV-G, resulted in optimal titers of NiV-pseudotyped particles (NiVpp) (∼10(6) IU/ml), even without ultracentrifugation. To further enhance the infectivity of NiVpp, we engineered a hyperfusogenic NiV-F protein lacking an N-linked glycosylation site (T5FΔN3). T5FΔN3/wt G particles exhibited enhanced infectivity on less permissive cell lines and efficiently targeted ephrinB2(+) cells even in a 1,000-fold excess of ephrinB2-negative cells, all without any loss of specificity, as entry was abrogated by soluble ephrinB2. NiVpp also transduced human embryonic, hematopoietic, and neural stem cell populations in an ephrinB2-dependent manner. Finally, intravenous administration of the luciferase reporter NiVpp-T5FΔN3/G to mice resulted in signals being detected in the spleen and lung but not in the liver. Bypassing the liver sink is a critical barrier for targeted gene therapy. The extraordinary specificity of NiV-G for ephrinB2 holds promise for targeting specific ephrinB2(+) populations in vivo or in vitro.
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http://dx.doi.org/10.1128/JVI.02032-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3571488PMC
February 2013

Characterization and therapeutic potential of induced pluripotent stem cell-derived cardiovascular progenitor cells.

PLoS One 2012 9;7(10):e45603. Epub 2012 Oct 9.

Department of Medicine and Physiology, Cardiovascular Research Laboratory, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.

Background: Cardiovascular progenitor cells (CPCs) have been identified within the developing mouse heart and differentiating pluripotent stem cells by intracellular transcription factors Nkx2.5 and Islet 1 (Isl1). Study of endogenous and induced pluripotent stem cell (iPSC)-derived CPCs has been limited due to the lack of specific cell surface markers to isolate them and conditions for their in vitro expansion that maintain their multipotency.

Methodology/principal Findings: We sought to identify specific cell surface markers that label endogenous embryonic CPCs and validated these markers in iPSC-derived Isl1(+)/Nkx2.5(+) CPCs. We developed conditions that allow propagation and characterization of endogenous and iPSC-derived Isl1(+)/Nkx2.5(+) CPCs and protocols for their clonal expansion in vitro and transplantation in vivo. Transcriptome analysis of CPCs from differentiating mouse embryonic stem cells identified a panel of surface markers. Comparison of these markers as well as previously described surface markers revealed the combination of Flt1(+)/Flt4(+) best identified and facilitated enrichment for Isl1(+)/Nkx2.5(+) CPCs from embryonic hearts and differentiating iPSCs. Endogenous mouse and iPSC-derived Flt1(+)/Flt4(+) CPCs differentiated into all three cardiovascular lineages in vitro. Flt1(+)/Flt4(+) CPCs transplanted into left ventricles demonstrated robust engraftment and differentiation into mature cardiomyocytes (CMs).

Conclusion/significance: The cell surface marker combination of Flt1 and Flt4 specifically identify and enrich for an endogenous and iPSC-derived Isl1(+)/Nkx2.5(+) CPC with trilineage cardiovascular potential in vitro and robust ability for engraftment and differentiation into morphologically and electrophysiologically mature adult CMs in vivo post transplantation into adult hearts.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0045603PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3467279PMC
April 2013

Combinatorial assembly of developmental stage-specific enhancers controls gene expression programs during human erythropoiesis.

Dev Cell 2012 Oct 4;23(4):796-811. Epub 2012 Oct 4.

Division of Hematology/Oncology, Children's Hospital Boston and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115, USA.

Gene-distal enhancers are critical for tissue-specific gene expression, but their genomic determinants within a specific lineage at different stages of development are unknown. Here we profile chromatin state maps, transcription factor occupancy, and gene expression profiles during human erythroid development at fetal and adult stages. Comparative analyses of human erythropoiesis identify developmental stage-specific enhancers as primary determinants of stage-specific gene expression programs. We find that erythroid master regulators GATA1 and TAL1 act cooperatively within active enhancers but confer little predictive value for stage specificity. Instead, a set of stage-specific coregulators collaborates with master regulators and contributes to differential gene expression. We further identify and validate IRF2, IRF6, and MYB as effectors of an adult-stage expression program. Thus, the combinatorial assembly of lineage-specific master regulators and transcriptional coregulators within developmental stage-specific enhancers determines gene expression programs and temporal regulation of transcriptional networks in a mammalian genome.
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http://dx.doi.org/10.1016/j.devcel.2012.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3477283PMC
October 2012

Dynamic distribution of linker histone H1.5 in cellular differentiation.

PLoS Genet 2012 30;8(8):e1002879. Epub 2012 Aug 30.

Department of Biological Chemistry, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.

Linker histones are essential components of chromatin, but the distributions and functions of many during cellular differentiation are not well understood. Here, we show that H1.5 binds to genic and intergenic regions, forming blocks of enrichment, in differentiated human cells from all three embryonic germ layers but not in embryonic stem cells. In differentiated cells, H1.5, but not H1.3, binds preferentially to genes that encode membrane and membrane-related proteins. Strikingly, 37% of H1.5 target genes belong to gene family clusters, groups of homologous genes that are located in proximity to each other on chromosomes. H1.5 binding is associated with gene repression and is required for SIRT1 binding, H3K9me2 enrichment, and chromatin compaction. Depletion of H1.5 results in loss of SIRT1 and H3K9me2, increased chromatin accessibility, deregulation of gene expression, and decreased cell growth. Our data reveal for the first time a specific and novel function for linker histone subtype H1.5 in maintenance of condensed chromatin at defined gene families in differentiated human cells.
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http://dx.doi.org/10.1371/journal.pgen.1002879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3431313PMC
January 2013

Lymphoid priming in human bone marrow begins before expression of CD10 with upregulation of L-selectin.

Nat Immunol 2012 Oct 2;13(10):963-71. Epub 2012 Sep 2.

Department of Pathology & Laboratory Medicine, University of California, Los Angeles, California, USA.

Expression of the cell-surface antigen CD10 has long been used to define the lymphoid commitment of human cells. Here we report a unique lymphoid-primed population in human bone marrow that was generated from hematopoietic stem cells (HSCs) before onset of the expression of CD10 and commitment to the B cell lineage. We identified this subset by high expression of the homing molecule L-selectin (CD62L). CD10(-)CD62L(hi) progenitors had full lymphoid and monocytic potential but lacked erythroid potential. Gene-expression profiling placed the CD10(-)CD62L(hi) population at an intermediate stage of differentiation between HSCs and lineage-negative (Lin(-)) CD34(+)CD10(+) progenitors. CD62L was expressed on immature thymocytes, and its ligands were expressed at the cortico-medullary junction of the thymus, which suggested a possible role for this molecule in homing to the thymus. Our studies identify the earliest stage of lymphoid priming in human bone marrow.
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http://dx.doi.org/10.1038/ni.2405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3448017PMC
October 2012

Scl represses cardiomyogenesis in prospective hemogenic endothelium and endocardium.

Cell 2012 Aug;150(3):590-605

Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA.

Endothelium in embryonic hematopoietic tissues generates hematopoietic stem/progenitor cells; however, it is unknown how its unique potential is specified. We show that transcription factor Scl/Tal1 is essential for both establishing the hematopoietic transcriptional program in hemogenic endothelium and preventing its misspecification to a cardiomyogenic fate. Scl(-/-) embryos activated a cardiac transcriptional program in yolk sac endothelium, leading to the emergence of CD31+Pdgfrα+ cardiogenic precursors that generated spontaneously beating cardiomyocytes. Ectopic cardiogenesis was also observed in Scl(-/-) hearts, where the disorganized endocardium precociously differentiated into cardiomyocytes. Induction of mosaic deletion of Scl in Scl(fl/fl)Rosa26Cre-ER(T2) embryos revealed a cell-intrinsic, temporal requirement for Scl to prevent cardiomyogenesis from endothelium. Scl(-/-) endothelium also upregulated the expression of Wnt antagonists, which promoted rapid cardiomyocyte differentiation of ectopic cardiogenic cells. These results reveal unexpected plasticity in embryonic endothelium such that loss of a single master regulator can induce ectopic cardiomyogenesis from endothelial cells.
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http://dx.doi.org/10.1016/j.cell.2012.06.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3624753PMC
August 2012

Trophoblasts regulate the placental hematopoietic niche through PDGF-B signaling.

Dev Cell 2012 Mar 1;22(3):651-9. Epub 2012 Mar 1.

Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.

The placenta is a hematopoietic organ that supports hematopoietic stem/progenitor cell (HSPC) generation and expansion without promoting differentiation. We identified PDGF-B signaling in trophoblasts as a key component of the unique placental hematopoietic microenvironment that protects HSPCs from premature differentiation. Loss of PDGF-B or its receptor, PDGFRβ, induced definitive erythropoiesis in placental labyrinth vasculature. This was evidenced by accumulation of CFU-Es and actively proliferating definitive erythroblasts that clustered around central macrophages, highly reminiscent of erythropoiesis in the fetal liver. Ectopic erythropoiesis was not due to a requirement of PDGF-B signaling in hematopoietic cells but rather in placental trophoblasts, which upregulated Epo in the absence of PDGF-B signaling. Furthermore, overexpression of hEPO specifically in the trophoblasts in vivo was sufficient to convert the placenta into an erythropoietic organ. These data provide genetic evidence of a signaling pathway that is required to restrict erythroid differentiation to specific anatomical niches during development.
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http://dx.doi.org/10.1016/j.devcel.2011.12.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3395466PMC
March 2012

Return to youth with Sox17.

Genes Dev 2011 Aug;25(15):1557-62

Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095, USA.

Maturation of hematopoietic stem cells (HSCs) from fetal to adult state and differentiation to progenitors are thought to follow a one-way street. In this issue of Genes & Development, He and colleagues (pp. 1613-1627) show that overexpression of Sox17 can convert adult multipotential progenitors to self-renewing HSCs that possess fetal properties. These findings challenge the irreversibility of hematopoietic development, and open up new perspectives to understand the different forms of HSC self-renewal at distinct stages of ontogeny and during transformation.
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http://dx.doi.org/10.1101/gad.17328611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182022PMC
August 2011

Regulated expression of microRNAs-126/126* inhibits erythropoiesis from human embryonic stem cells.

Blood 2011 Feb 16;117(7):2157-65. Epub 2010 Dec 16.

Howard Hughes Medical Institute, Los Angeles, CA, USA.

MicroRNAs (miRs) play an important role in cell differentiation and maintenance of cell identity, but relatively little is known of their functional role in modulating human hematopoietic lineage differentiation. Human embryonic stem cells (hESCs) provide a model system to study early human hematopoiesis. We differentiated hESCs by embryoid body (EB) formation and compared the miR expression profile of undifferentiated hESCs to CD34(+) EB cells. miRs-126/126* were the most enriched of the 7 miRs that were up-regulated in CD34(+) cells, and their expression paralleled the kinetics of hematopoietic transcription factors RUNX1, SCL, and PU.1. To define the role of miRs-126/126* in hematopoiesis, we created hESCs overexpressing doxycycline-regulated miRs-126/126* and analyzed their hematopoietic differentiation. Induction of miRs-126/126* during both EB differentiation and colony formation reduced the number of erythroid colonies, suggesting an inhibitory role of miRs-126/126* in erythropoiesis. Protein tyrosine phosphatase, nonreceptor type 9 (PTPN9), a protein tyrosine phosphatase that is required for growth and expansion of erythroid cells, is one target of miR-126. PTPN9 restoration partially relieved the suppressed erythropoiesis caused by miRs-126/126*. Our results define an important function of miRs-126/126* in negative regulation of erythropoiesis, providing the first evidence for a role of miR in hematopoietic differentiation of hESCs.
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http://dx.doi.org/10.1182/blood-2010-08-302711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3062325PMC
February 2011

Hematopoietic stem cell development in the placenta.

Int J Dev Biol 2010 ;54(6-7):1089-98

University of California Los Angeles, Los Angeles, CA 90095, USA.

The placenta is a highly vascularized organ that mediates fetal-maternal exchange during pregnancy and is thereby vital for the survival and growth of the developing embryo. In addition to having this well-established role in supporting pregnancy, the placenta was recently shown to function as a hematopoietic organ. The placenta is unique among other fetal hematopoietic organs, as it is capable of both generating multipotential hematopoietic cells de novo and establishing a major hematopoietic stem cell (HSC) pool in the conceptus, while protecting HSCs from premature differentiation. The mouse placenta contains two distinct vascular regions that support hematopoiesis: the large vessels in the chorionic plate where HSCs/progenitors are thought to emerge and the labyrinth vasculature where nascent HSCs/progenitors may colonize for expansion and possible functional maturation. Defining how this cytokine- and growth factor rich organ supports HSC generation, maturation and expansion may ultimately help to establish culture protocols for HSC expansion or de novo generation from pluripotent cells.
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http://dx.doi.org/10.1387/ijdb.103070cgDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4285582PMC
February 2011