Generation of functionally distinct hemogenic endothelial cell populations from pluripotent stem cells.

A diverse array of protocols have been established for the directed differentiation of human pluripotent stem cells (hPSCs) into a variety of cell types, including blood cells, for modeling development and disease, and for the development of cell-based therapeutics. These protocols recapitulate various signaling requirements essential for the establishment of the hematopoietic systems during embryonic development. However, in many instances, the functional properties of those progenitors, and their relevance to human development, remains unclear. The human embryo, much like other vertebrate model organisms, generates hematopoietic cells via successive anatomical location- and time-specific waves, each yielding cells with distinct functional and molecular characteristics. Each of these progenitor "waves" is characterized at the time of emergence of the direct hematopoietic progenitor in the vasculature, the hemogenic endothelial cell (HEC). Critically, despite decades of study in model organisms, the origins of each of these HEC populations remain unclear. Fortunately, through the directed differentiation of hPSCs, recent insights have been made into the earliest origins of each HEC population, revealing that each arises from transcriptionally and phenotypically distinct subsets of nascent mesoderm. Here, we outline the protocols to generate each mesodermal and HEC population via the formation of embryoid bodies and subsequent stage-specific signal manipulation. Through implementation of these discrete signal manipulations, it is possible to obtain human HEC populations that are exclusively extraembryonic-like or exclusively intraembryonic-like, enabling comparative developmental biology studies or specific translational applications.

Vade-MECOM: How to peel back the layers of hematopoiesis.

A recent study1 demonstrates how hematopoietic stem cells (HSCs) contribute minimally to blood and immune cell production during development and only become active postnatally. The work also reveals how Mecom expression can be used to distinguish rare HSCs from the more abundant progenitors that arise to maintain embryonic hematopoiesis.

Cellular and transcriptional dynamics of human neutrophils at steady state and upon stress.

Traditionally viewed as poorly plastic, neutrophils are now recognized as functionally diverse; however, the extent and determinants of neutrophil heterogeneity in humans remain unclear. We performed a comprehensive immunophenotypic and transcriptome analysis, at a bulk and single-cell level, of neutrophils from healthy donors and patients undergoing stress myelopoiesis upon exposure to growth factors, transplantation of hematopoietic stem cells (HSC-T), development of pancreatic cancer and viral infection. We uncover an extreme diversity of human neutrophils in vivo, reflecting the rates of cell mobilization, differentiation and exposure to environmental signals. Integrated control of developmental and inducible transcriptional programs linked flexible granulopoietic outputs with elicitation of stimulus-specific functional responses. In this context, we detected an acute interferon (IFN) response in the blood of patients receiving HSC-T that was mirrored by marked upregulation of IFN-stimulated genes in neutrophils but not in monocytes. Systematic characterization of human neutrophil plasticity may uncover clinically relevant biomarkers and support the development of diagnostic and therapeutic tools.

Modeling human yolk sac hematopoiesis with pluripotent stem cells.

In the mouse, the first hematopoietic cells are generated in the yolk sac from the primitive, erythro-myeloid progenitor (EMP) and lymphoid programs that are specified before the emergence of hematopoietic stem cells. While many of the yolk sac-derived populations are transient, specific immune cell progeny seed developing tissues, where they function into adult life. To access the human equivalent of these lineages, we modeled yolk sac hematopoietic development using pluripotent stem cell differentiation. Here, we show that the combination of Activin A, BMP4, and FGF2 induces a population of KDR+CD235a/b+ mesoderm that gives rise to the spectrum of erythroid, myeloid, and T lymphoid lineages characteristic of the mouse yolk sac hematopoietic programs, including the Vδ2+ subset of γ/δ T cells that develops early in the human embryo. Through clonal analyses, we identified a multipotent hematopoietic progenitor with erythroid, myeloid, and T lymphoid potential, suggesting that the yolk sac EMP and lymphoid lineages may develop from a common progenitor.

B1 lymphocytes develop independently of Notch signaling during mouse embryonic development.

B1 lymphocytes are a small but unique component of the innate immune-like cells. However, their ontogenic origin is still a matter of debate. Although it is widely accepted that B1 cells originate early in fetal life, whether or not they arise from hematopoietic stem cells (HSCs) is still unclear. In order to shed light on the B1 cell origin, we set out to determine whether their lineage specification is dependent on Notch signaling, which is essential for the HSC generation and, therefore, all derivatives lineages. Using mouse embryonic stem cells (mESCs) to recapitulate murine embryonic development, we have studied the requirement for Notch signaling during the earliest B-cell lymphopoiesis and found that Rbpj-deficient mESCs are able to generate B1 cells. Their Notch independence was confirmed in ex vivo experiments using Rbpj-deficient embryos. In addition, we found that upregulation of Notch signaling induced the emergence of B2 lymphoid cells. Taken together, these findings indicate that control of Notch signaling dose is crucial for different B-cell lineage specification from endothelial cells and provides pivotal information for their in vitro generation from PSCs for therapeutic applications. This article has an associated 'The people behind the papers' interview.

Haematopoietic stem and progenitor cells from human pluripotent stem cells.

A variety of tissue lineages can be differentiated from pluripotent stem cells by mimicking embryonic development through stepwise exposure to morphogens, or by conversion of one differentiated cell type into another by enforced expression of master transcription factors. Here, to yield functional human haematopoietic stem cells, we perform morphogen-directed differentiation of human pluripotent stem cells into haemogenic endothelium followed by screening of 26 candidate haematopoietic stem-cell-specifying transcription factors for their capacity to promote multi-lineage haematopoietic engraftment in mouse hosts. We recover seven transcription factors (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1 and SPI1) that are sufficient to convert haemogenic endothelium into haematopoietic stem and progenitor cells that engraft myeloid, B and T cells in primary and secondary mouse recipients. Our combined approach of morphogen-driven differentiation and transcription-factor-mediated cell fate conversion produces haematopoietic stem and progenitor cells from pluripotent stem cells and holds promise for modelling haematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders.

Directed differentiation of definitive hemogenic endothelium and hematopoietic progenitors from human pluripotent stem cells.

The generation of hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) remains a major goal for regenerative medicine and disease modeling. However, hPSC differentiation cultures produce mostly hematopoietic progenitors belonging to the embryonic HSC-independent hematopoietic program, which may not be relevant or accurate for modeling normal and disease-state adult hematopoietic processes. Through a stage-specific directed differentiation approach, it is now possible to generate exclusively definitive hematopoietic progenitors from hPSCs showing characteristics of the more developmentally advanced fetal hematopoiesis. Here, we summarize recent efforts at generating hPSC-derived definitive hematopoiesis through embryoid body differentiation under defined conditions. Embryoid bodies are generated through enzymatic dissociation of hPSCs from matrigel-coated plasticware, followed by recombinant BMP4, driving mesoderm specification. Definitive hematopoiesis is specified by a GSK3ß-inhibitor, followed by recombinant VEGF and supportive hematopoietic cytokines. The CD34+ cells obtained using this method are then suitable for hematopoietic assays for definitive hematopoietic potential.

Human definitive haemogenic endothelium and arterial vascular endothelium represent distinct lineages.

The generation of haematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) will depend on the accurate recapitulation of embryonic haematopoiesis. In the early embryo, HSCs develop from the haemogenic endothelium (HE) and are specified in a Notch-dependent manner through a process named endothelial-to-haematopoietic transition (EHT). As HE is associated with arteries, it is assumed that it represents a subpopulation of arterial vascular endothelium (VE). Here we demonstrate at a clonal level that hPSC-derived HE and VE represent separate lineages. HE is restricted to the CD34(+)CD73(-)CD184(-) fraction of day 8 embryoid bodies and it undergoes a NOTCH-dependent EHT to generate RUNX1C(+) cells with multilineage potential. Arterial and venous VE progenitors, in contrast, segregate to the CD34(+)CD73(med)CD184(+) and CD34(+)CD73(hi)CD184(-) fractions, respectively. Together, these findings identify HE as distinct from VE and provide a platform for defining the signalling pathways that regulate their specification to functional HSCs.

Wnt signaling controls the specification of definitive and primitive hematopoiesis from human pluripotent stem cells.

Efforts to derive hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) are complicated by the fact that embryonic hematopoiesis consists of two programs, primitive and definitive, that differ in developmental potential. As only definitive hematopoiesis generates HSCs, understanding how this program develops is essential for being able to produce this cell population in vitro. Here we show that both hematopoietic programs transition through hemogenic endothelial intermediates and develop from KDR(+)CD34(-)CD144(-) progenitors that are distinguished by CD235a expression. Generation of primitive progenitors (KDR(+)CD235a(+)) depends on stage-specific activin-nodal signaling and inhibition of the Wnt-ß-catenin pathway, whereas specification of definitive progenitors (KDR(+)CD235a(-)) requires Wnt-ß-catenin signaling during this same time frame. Together, these findings establish simple selective differentiation strategies for the generation of primitive or definitive hematopoietic progenitors by Wnt-ß-catenin manipulation, and in doing so provide access to enriched populations for future studies on hPSC-derived hematopoietic development.

Retinoic acid signaling is essential for embryonic hematopoietic stem cell development.

Hematopoietic stem cells (HSCs) develop from a specialized subpopulation of endothelial cells known as hemogenic endothelium (HE). Although the HE origin of HSCs is now well established in different species, the signaling pathways that control this transition remain poorly understood. Here, we show that activation of retinoic acid (RA) signaling in aorta-gonad-mesonephros-derived HE ex vivo dramatically enhanced its HSC potential, whereas conditional inactivation of the RA metabolizing enzyme retinal dehydrogenase 2 in VE-cadherin expressing endothelial cells in vivo abrogated HSC development. Wnt signaling completely blocked the HSC inductive effects of RA modulators, whereas inhibition of the pathway promoted the development of HSCs in the absence of RA signaling. Collectively, these findings position RA and Wnt signaling as key regulators of HSC development and in doing so provide molecular insights that will aid in developing strategies for their generation from pluripotent stem cells.

T lymphocyte potential marks the emergence of definitive hematopoietic progenitors in human pluripotent stem cell differentiation cultures.

The efficient generation of hematopoietic stem cells from human pluripotent stem cells is dependent on the appropriate specification of the definitive hematopoietic program during differentiation. In this study, we used T lymphocyte potential to track the onset of definitive hematopoiesis from human embryonic and induced pluripotent stem cells differentiated with specific morphogens in serum- and stromal-free cultures. We show that this program develops from a progenitor population with characteristics of hemogenic endothelium, including the expression of CD34, VE-cadherin, GATA2, LMO2, and RUNX1. Along with T cells, these progenitors display the capacity to generate myeloid and erythroid cells. Manipulation of Activin/Nodal signaling during early stages of differentiation revealed that development of the definitive hematopoietic progenitor population is not dependent on this pathway, distinguishing it from primitive hematopoiesis. Collectively, these findings demonstrate that it is possible to generate T lymphoid progenitors from pluripotent stem cells and that this lineage develops from a population whose emergence marks the onset of human definitive hematopoiesis.

Primitive erythropoiesis is regulated by miR-126 via nonhematopoietic Vcam-1+ cells.

Primitive erythropoiesis defines the onset of hematopoiesis in the yolk sac of the early embryo and is initiated by the emergence of progenitors assayed as colony-forming cells (EryP-CFCs). EryP-CFCs are detected for only a narrow window during embryonic development, suggesting that both their initiation and termination are tightly controlled. Using the embryonic stem differentiation system to model primitive erythropoiesis, we found that miR-126 regulates the termination of EryP-CFC development. Analyses of miR-126 null embryos revealed that this miR also regulates EryP-CFCs in vivo. We identified vascular cell adhesion molecule-1 (Vcam-1) expressed by a mesenchymal cell population as a relevant target of miR-126. Interaction of EryP-CFCs with Vcam-1 accelerated their maturation to ßh1-globin(+) and Ter119(+) cells through a Src family kinase. These findings uncover a cell nonautonomous regulatory pathway for primitive erythropoiesis that may provide insight into the mechanism(s) controlling the developmental switch from primitive to definitive hematopoiesis.

Clonal characterization of rat muscle satellite cells: proliferation, metabolism and differentiation define an intrinsic heterogeneity.

Satellite cells (SCs) represent a distinct lineage of myogenic progenitors responsible for the postnatal growth, repair and maintenance of skeletal muscle. Distinguished on the basis of their unique position in mature skeletal muscle, SCs were considered unipotent stem cells with the ability of generating a unique specialized phenotype. Subsequently, it was demonstrated in mice that opposite differentiation towards osteogenic and adipogenic pathways was also possible. Even though the pool of SCs is accepted as the major, and possibly the only, source of myonuclei in postnatal muscle, it is likely that SCs are not all multipotent stem cells and evidences for diversities within the myogenic compartment have been described both in vitro and in vivo. Here, by isolating single fibers from rat flexor digitorum brevis (FDB) muscle we were able to identify and clonally characterize two main subpopulations of SCs: the low proliferative clones (LPC) present in major proportion (approximately 75%) and the high proliferative clones (HPC), present instead in minor amount (approximately 25%). LPC spontaneously generate myotubes whilst HPC differentiate into adipocytes even though they may skip the adipogenic program if co-cultured with LPC. LPC and HPC differ also for mitochondrial membrane potential (DeltaPsi(m)), ATP balance and Reactive Oxygen Species (ROS) generation underlying diversities in metabolism that precede differentiation. Notably, SCs heterogeneity is retained in vivo. SCs may therefore be comprised of two distinct, though not irreversibly committed, populations of cells distinguishable for prominent differences in basal biological features such as proliferation, metabolism and differentiation. By these means, novel insights on SCs heterogeneity are provided and evidences for biological readouts potentially relevant for diagnostic purposes described.

Human and murine amniotic fluid c-Kit+Lin- cells display hematopoietic activity.

We have isolated c-Kit(+)Lin(-) cells from both human and murine amniotic fluid (AF) and investigated their hematopoietic potential. In vitro, the c-Kit(+)Lin(-) population in both species displayed a multilineage hematopoietic potential, as demonstrated by the generation of erythroid, myeloid, and lymphoid cells. In vivo, cells belonging to all 3 hematopoietic lineages were found after primary and secondary transplantation of murine c-Kit(+)Lin(-) cells into immunocompromised hosts, thus demonstrating the ability of these cells to self-renew. Gene expression analysis of c-Kit(+) cells isolated from murine AF confirmed these results. The presence of cells with similar characteristics in the surrounding amnion indicates the possible origin of AF c-Kit(+)Lin(-) cells. This is the first report showing that cells isolated from the AF do have hematopoietic potential; our results support the idea that AF may be a new source of stem cells for therapeutic applications.

Mesenchymal stromal cells can be derived from bone marrow CD133+ cells: implications for therapy.

It is known that the bone marrow (BM) CD133(+) cells play an important role in the hematopoietic compartment, but this is not their only role. The cells indeed can take part in vascular reconstitution when they become endothelial cells (EC), in skeletal muscle fiber regeneration when there is a switch in muscle precursors, and to cardiomyocyte phenotypic conversion when differentiating in cardiomyocytes-like cells. While the role in hematopoiesis and vasculogenesis of the selected cells is well established, their ability to differentiate along multiple non-EC lineages has not yet been fully elucidated. The goal of this study is to assert whether human CD133(+)BM-derived cells are able to differentiate in vitro, besides to blood cells, cell lineages pertinent to the mesoderm germ layers. To this end, we isolated CD133(+) cells using a clinically approved methodology and compared their differentiation potential to that of hematopoietic progenitor cells (HPCs) and mesenchymal stem cells (MSCs) obtained from the same BM samples. In our culture conditions, CD133 expression was consistently decreased after passage 2, as well as the expression of the stemness markers c-kit and OCT4, whereas expression of Stage Specific Embryonic Antigen 4 (SSEA4) remained consistent in all different conditions. Expanded CD133 were also positive for HLA-ABC, but negative for HLA-DR, in accordance with what has been previously reported for MSCs. Moreover, CD133(+) cells from human BM demonstrated a wide range of differentiation potential, encompassing not only mesodermal but also ectodermal (neurogenic) cell lineages. CD133 antigen could be potentially used to select a cell population with similar characteristics as MSCs for therapeutic applications.

Human adenylate kinase 2 deficiency causes a profound hematopoietic defect associated with sensorineural deafness.

Reticular dysgenesis is an autosomal recessive form of human severe combined immunodeficiency characterized by an early differentiation arrest in the myeloid lineage and impaired lymphoid maturation. In addition, affected newborns have bilateral sensorineural deafness. Here we identify biallelic mutations in AK2 (adenylate kinase 2) in seven individuals affected with reticular dysgenesis. These mutations result in absent or strongly decreased protein expression. We then demonstrate that restoration of AK2 expression in the bone marrow cells of individuals with reticular dysgenesis overcomes the neutrophil differentiation arrest, underlining its specific requirement in the development of a restricted set of hematopoietic lineages. Last, we establish that AK2 is specifically expressed in the stria vascularis region of the inner ear, which provides an explanation of the sensorineural deafness in these individuals. These results identify a previously unknown mechanism involved in regulation of hematopoietic cell differentiation and in one of the most severe human immunodeficiency syndromes.

In vivo delivery of naked antisense oligos in aged mdx mice: analysis of dystrophin restoration in skeletal and cardiac muscle.

Antisense-mediated exon skipping holds great potential for the treatment of DMD. In mdx mice, functional recovery of skeletal muscle has been obtained upon systemic delivery of "naked" oligonucleotides or viral vectors encoding for antisense snRNAs. However, amongst the studies reported so far, which used either neonatal or young adult animals--only one achieved dystrophin restoration in cardiac muscle, using an adeno-associated vector. Here we report the in vivo delivery of morpholino oligos in aged mdx mice, both in skeletal muscle, via intra-arterial injection, and in cardiac muscle, via intra-muscular injection. Localized intra-arterial delivery yielded high levels of dystrophin restoration and just two doses of 100 microg each resulted into detectable force recovery in the EDL muscles of treated limbs. On the other hand, upon intra-cardiac injections in the left ventricle wall the skipping effect was much lower than what obtained in tibialis anterior muscles injected with comparable amounts of oligos. This latter finding suggests that even upon direct delivery antisense-mediated dystrophin restoration in cardiac muscle might suffer from limitations that do not exist in skeletal muscle.

High transduction efficiency of human amniotic fluid stem cells mediated by adenovirus vectors.

In the last few years some studies have shown the possibility of deriving progenitors with various potential from the amniotic fluid. Amniocentesis is a widely accepted method for prenatal diagnosis; it is associated with low risk both for the mother and the fetus and overcomes the ethical problems commonly associated to other sources. Recently we have described that amniotic fluid stem (AFS) cells, for their ability to differentiate to various lineages, could represent a good candidate for therapeutic applications. For gene therapy purposes human AFS (hAFS) cells should be genetically modified with a therapeutic gene and delivered systematically or injected directly into the tissue of interest. The aim of this study was to investigate the feasibility of transducing hAFS cells with adenoviral vectors and to determine whether transduced stem cells retain the ability to differentiate into different lineages. Herein, we showed that hAFS cells could be efficiently infected by first generation adenovirus vectors. In addition, we demonstrated that infection and expression of two different marker genes, LacZ and EGFP, have no effect on cells phenotype and differentiation potential. In particular, on undifferentiated status, hAFS cells continued to express both the transgenes and stemness cell markers OCT4 and SSEA4. When cultured under mesenchymal conditions, infected cells could still differentiate into osteocytes and adipocytes expressing lineage specific genes. These preliminary findings suggest that adenovirus may be useful to engineer populations of pluripotent stem cells, which may be used in a wide range of gene therapy treatments.

Regulatory mutations in transforming growth factor-beta3 gene cause arrhythmogenic right ventricular cardiomyopathy type 1.

OBJECTIVE: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetically heterogeneous disorder characterized by fibro-fatty replacement of the right ventricular myocardium, associated with high risk of sudden death. The objective of this study is to identify the gene involved in ARVD1, which has been elusive ever since its locus was mapped to chromosome 14q24.3. METHODS AND RESULTS: Mutation screening of the promoter and untranslated regions (UTRs) of the transforming growth factor-beta3 (TGFbeta3) gene was performed by direct sequencing of genomic DNA of one index case belonging to an ARVD1 family including 38 members in four generations. We detected a nucleotide substitution (c.-36G>A) in 5' UTR of TGFbeta3 gene, invariably associated with the typical ARVC clinical phenotype in the affected family members, according to the established diagnostic criteria. Investigation extended to 30 unrelated ARVC patients, performed by denaturing high-performance liquid chromatography (DHPLC), led to the identification of an additional mutation (c.1723C>T) in the 3' UTR of one proband. Neither nucleotide change was found in 300 control subjects. In vitro expression assays with constructs containing the mutations showed that mutated UTRs were twofold more active than wild-types. CONCLUSION: We identified TGFbeta3 as the disease gene involved in ARVD1. The identification of a novel ARVC gene will increase the power of the genetic screening for early diagnosis of asymptomatic carriers among relatives of ARVC patients.