Hanna Mikkola

Human Fetal Hemoglobin Expression Is Regulated by the Developmental Stage- Specific Repressor BCL11A

Differences in the amount of fetal hemoglobin (HbF) that persists into adulthood affect the severity of sickle cell disease and the β-thalassemia syndromes. Genetic association studies have identified sequence variants in the gene BCL11A that influence HbF levels. Here, we examine BCL11A as a potential regulator of HbF expression. The high-HbF BCL11A genotype is associated with reduced BCL11A expression. Moreover, abundant expression of full-length forms of BCL11A is developmentally restricted to adult erythroid cells. Down-regulation of BCL11A expression in primary adult erythroid cells leads to robust HbF expression. Consistent with a direct role of BCL11A in globin gene regulation, we find that BCL11A occupies several discrete sites in the β-globin gene cluster. BCL11A emerges as a therapeutic target for reactivation of HbF in β-hemoglobin disorders.

The journey of developing hematopoietic stem cells

Hematopoietic stem cells (HSCs) develop during embryogenesis in a complex process that involves multiple anatomical sites. Once HSC precursors have been specified from mesoderm, they have to mature into functional HSCs and undergo self-renewing divisions to generate a pool of HSCs. During this process, developing HSCs migrate through various embryonic niches, which provide signals for their establishment and the conservation of their self-renewal ability. These processes have to be recapitulated to generate HSCs from embryonic stem cells. Elucidating the interactions between developing HSCs and their niches should facilitate the generation and expansion of HSCs in vitro to exploit their clinical potential.

Haematopoietic stem cells retain long-term repopulating activity and multipotency in the absence of stem-cell leukaemia SCL/tal-1 gene.

The production of blood cells is sustained throughout the lifetime of an individual by haematopoietic stem cells (HSCs). Specification of HSCs from mesoderm during embryonic development requires the stem cell leukaemia SCL/tal-1 gene product. Forced expression of SCL/tal-1 strongly induces blood formation in embryos, indicating that this gene has a dominant role in commitment to haematopoiesis. In the adult haematopoietic system, expression of SCL/tal-1 is enriched in HSCs and multipotent progenitors, and in erythroid and megakaryocytic lineages, consistent with roles for this factor in adult haematopoiesis. Here we assess by conditional gene targeting whether SCL/tal-1 is required continuously for the identity and function of HSCs. We find that SCL/tal-1 is dispensable for HSC engraftment, self-renewal and differentiation into myeloid and lymphoid lineages; however, the proper differentiation of erythroid and megakaryocytic precursors is dependent on SCL/tal-1. Thus, SCL/tal-1 is essential for the genesis of HSCs, but its continued expression is not essential for HSC functions. These findings contrast with lineage choice mechanisms, in which the identity of haematopoietic lineages requires continuous transcription factor expression.

Reprogrammed Mouse Fibroblasts Differentiate into Cells of the Cardiovascular and Hematopoietic Lineages

Forced expression of the four transcription factors Oct4, Sox2, c‐Myc, and Klf4 is sufficient to confer a pluripotent state upon the murine fibroblast genome, generating induced pluripotent stem (iPS) cells. Although the differentiation potential of these cells is thought to be equivalent to that of embryonic stem (ES) cells, it has not been rigorously determined. In this study, we sought to identify the capacity of iPS cells to differentiate into Flk1‐positive progenitors and their mesodermal progeny, including cells of the cardiovascular and hematopoietic lineages. Immunostaining of tissues from iPS cell‐derived chimeric mice demonstrated that iPS cells could contribute in vivo to cardiomyocytes, smooth muscle cells, endothelial cells, and hematopoietic cells. To compare the in vitro differentiation potential of murine ES and iPS cells, we either induced embryoid body (EB) formation of each cell type or cultured the cells on collagen type IV (ColIV), an extracellular matrix protein that had been reported to direct murine ES cell differentiation to mesodermal lineages. EB formation and exposure to ColIV both induced iPS cell differentiation into cells that expressed cardiovascular and hematopoietic markers. To determine whether ColIV‐differentiated iPS cells contained a progenitor cell with cardiovascular and hematopoietic differentiation potential, Flk1‐positive cells were isolated by magnetic cell sorting and exposed to specific differentiation conditions, which induced differentiation into functional cardiomyocytes, smooth muscle cells, endothelial cells, and hematopoietic cells. Our data demonstrate that murine iPS cells, like ES cells, can differentiate into cells of the cardiovascular and hematopoietic lineages and therefore may represent a valuable cell source for applications in regenerative medicine.

The Emergence of Hematopoietic Stem Cells Is Initiated in the Placental Vasculature in the Absence of Circulation

The mouse placenta was unveiled as an important reservoir for hematopoietic stem cells (HSCs), yet the origin of placental HSCs was unknown. By tracking developing HSCs by expression of Runx1-lacZ and CD41, we have found that HSCs emerge in large vessels in the placenta. Analysis of Ncx1(-/-) embryos, which lack a heartbeat, verified that HSC development is initiated in the placental vasculature independent of blood flow. However, fewer CD41+ hematopoietic cells were found in Ncx1(-/-) placentas than in controls, implying that some HSCs/progenitors colonize the placenta via circulation and/or HSC emergence is compromised without blood flow. Importantly, placentas from Ncx1(-/-) embryos possessed equal potential to generate myelo-erythroid and B and T lymphoid cells upon explant culture, verifying intact multilineage hematopoietic potential, characteristic of developing HSCs. These data suggest that, in addition to providing a niche for a large pool of HSCs prior to liver colonization, the placenta is a true site of HSC generation.

Derivation of Primordial Germ Cells from Human Embryonic and Induced Pluripotent Stem Cells Is Significantly Improved by Coculture with Human Fetal Gonadal Cells

The derivation of germ cells from human embryonic stem cells (hESCs) or human induced pluripotent stem (hIPS) cells represents a desirable experimental model and potential strategy for treating infertility. In the current study, we developed a triple biomarker assay for identifying and isolating human primordial germ cells (PGCs) by first evaluating human PGC formation during the first trimester in vivo. Next, we applied this technology to characterizing in vitro derived PGCs (iPGCs) from pluripotent cells. Our results show that codifferentiation of hESCs on human fetal gonadal stromal cells significantly improves the efficiency of generating iPGCs. Furthermore, the efficiency was comparable between various pluripotent cell lines regardless of origin from the inner cell mass of human blastocysts (hESCs), or reprogramming of human skin fibroblasts (hIPS). To better characterize the iPGCs, we performed Real‐time polymerase chain reaction, microarray, and bisulfite sequencing. Our results show that iPGCs at day 7 of differentiation are transcriptionally distinct from the somatic cells, expressing genes associated with pluripotency and germ cell development while repressing genes associated with somatic differentiation (specifically multiple HOX genes). Using bisulfite sequencing, we show that iPGCs initiate imprint erasure from differentially methylated imprinted regions by day 7 of differentiation. However, iPGCs derived from hIPS cells do not initiate imprint erasure as efficiently. In conclusion, our results indicate that triple positive iPGCs derived from pluripotent cells differentiated on hFGS cells correspond to committed first trimester germ cells (before 9 weeks) that have initiated the process of imprint erasure. STEM CELLS 2009;27:783–795

Novel Aspects of Blood Coagulation Factor XIII. I. Structure, Distribution, Activation, and Function

Blood coagulation factor XIII (FXIII) is a protransglutaminase that becomes activated by the concerted action of thrombin and Ca2+ in the final stage of the clotting cascade. In addition to plasma, FXIII also occurs in platelets, monocytes, and monocyte-derived macrophages. While the plasma factor is a heterotetramer consisting of paired A and B subunits (A2B2), its cellular counterpart lacks the B subunits and is a homodimer of potentially active A subunits (A2). The gene coding for the A and B subunits has been localized to chromosomes 6p24-25 and 1q31-32.1, respectively. The genomic as well as the primary protein structure of both subunits has been established, and most recently the three-dimensional structure of recombinant cellular FXIII has also been revealed. Monocytes/macrophages synthesize their own FXIII, and very likely FXIII in platelets is synthesized by the megakaryocytes. Cells of bone marrow origin seem to be the primary site for the synthesis of subunit A in plasma FXIII, but hepatocytes might also contribute. The B subunit of plasma FXIII is synthesized in the liver. Plasma FXIII circulates in association with its substrate precursor, fibrinogen. Fibrin(ogen) has an important regulatory role in the activation of plasma FXIII. The most important steps of the activation of plasma FXIII are the proteolytic removal of activation peptide by thrombin, the dissociation of subunits A and B, and the exposure of the originally buried active site on the free A subunits. The end result of this process is the formation of an active transglutaminase, which cross-links peptide chains through epsilon(gamma-glutamyl)lysyl isopeptide bonds. Cellular FXIII in platelets becomes activated through a nonproteolytic process. When intracytoplasmic Ca2+ is raised during platelet activation, the zymogen--in the absence of subunit B--assumes an active configuration. The protein substrates of activated FXIII include components of the clotting-fibrinolytic system, adhesive and contractile proteins. The main physiological function of plasma FXIII is to cross-link fibrin and protect it from the fibrinolytic plasmin. The latter effect is achieved mainly by covalently linking alpha 2 antiplasmin, the most potent physiological inhibitor of plasmin, to fibrin. Plasma FXIII seems to be involved in wound healing and tissue repair, and it is essential to maintaining pregnancy. Cellular FXIII, if exposed to the surface of the cells, might support or perhaps take over the hemostatic functions of plasma FXIII; however, its intracellular role has remained mostly unexplored.

Lentivirus vector gene expression during ES cell-derived hematopoietic development in vitro.

ABSTRACT The murine embryonal stem (ES) cell virus (MESV) can express transgenes from the long terminal repeat (LTR) promoter/enhancer in undifferentiated ES cells, but expression is turned off upon differentiation to embryoid bodies (EBs) and hematopoietic cells in vitro. We examined whether a human immunodeficiency virus type 1-based lentivirus vector pseudotyped with the vesicular stomatitis virus G protein (VSV-G) could transduce ES cells efficiently and express the green fluorescent protein (GFP) transgene from an internal phosphoglycerate kinase (PGK) promoter throughout development to hematopoietic cells in vitro. An oncoretrovirus vector containing the MESV LTR and the GFP gene was used for comparison. Fluorescence-activated cell sorting analysis of transduced CCE ES cells showed 99.8 and 86.7% GPF-expressing ES cells in the VSV-G-pseudotyped lentivirus (multiplicity of infection [MOI] = 59)- and oncoretrovirus (MOI = 590)-transduced cells, respectively. Therefore, VSV-G pseudotyping of lentiviral and oncoretrovirus vectors leads to efficient transduction of ES cells. Lentivirus vector integration was verified in the ES cell colonies by Southern blot analysis. When the transduced ES cells were differentiated in vitro, expression from the oncoretrovirus LTR was severely reduced or extinct in day 6 EBs and ES cell-derived hematopoietic colonies. In contrast, many lentivirus-transduced colonies, expressing the GFP gene in the undifferentiated state, continued to express the transgene throughout in vitro development to EBs at day 6, and many continued to express in cells derived from hematopoietic colonies. This experimental system can be used to analyze lentivirus vector design for optimal expression in hematopoietic cells and for gain-of-function experiments during ES cell development in vitro.

Association of FXIII Val34Leu with decreased risk of myocardial infarction in Finnish males

Factor XIII is a transglutaminase that crosslinks fibrin in the last steps of the coagulation process. A few polymorphic sites have been identified in this gene, one of them being a point mutation (FXIII Val34Leu), leading to an amino acid change of valine to leucine. Recently, in British patients, FXIII 34Leu allele was suggested to be associated with a decreased incidence of myocardial infarction (MI). PAI-1 4G/4G genotype seemed to lessen the beneficial effect of FXIII 34Leu allele. The aim of our study was to further investigate the possible protective role of the FXIII 34Leu allele against MI and its suggested interaction with the PAI-1 4G/5G polymorphism. We carried out genotype analyses for FXIII Val34Leu using solid-phase minisequencing in two independent Finnish study groups. In our study, the FXIII 34Leu allele was associated with a lower risk of MI (P = 0.009), however, the PAI-1 4G allele showed no interaction with this polymorphism. To establish the population frequency of the FXIII 34Leu allele and to study the possible variations in Finland four DNA pools from different geographical areas of Finland were genotyped. No significant differences in the allele frequencies were observed (21-28%) except in the Eastern Kainuu area (13%), an area with an increased risk of mortality from coronary artery disease (CAD), supporting the results presented above. The association of FXIII 34Leu variant with a lower incidence of myocardial infarction suggests a new role for FXIII in a polygenic thrombotic disease.

Combinatorial Assembly of Developmental Stage-Specific Enhancers Controls Gene Expression Programs during Human Erythropoiesis

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.