Cell therapy of primary myopathies

Mesoangioblasts are multipotent progenitors of mesodermal tissues. In vitro mesoangioblasts differentiate into many mesoderm cell types, such as smooth, cardiac and striated muscle, bone and endothelium. After transplantation mesoangioblasts colonize mostly mesoderm tissues and differentiate into many cell types of the mesoderm. When delivered through the arterial circulation, mesoangioblasts significantly restore skeletal muscle structure and function in a mouse model of muscular dystrophy. Their ability to extensively self-renew in vitro, while retaining multipotency, qualifies mesoangioblasts as a novel class of stem cells. Phenotype, properties and possible origin of mesoangioblasts are addressed in the first part of this paper. In the second part we will focus on the cell therapy approach for the treatment of Muscular Dystrophy and we will describe why mesangioblasts appear to be promising candidates for this strategy.     

Bidirectional induction toward paraxial mesodermal derivatives from mouse ES cells in chemically defined medium

Embryonic stem cells (ESCs) are a renewable cell source of tissue for regenerative therapies. The addition of bone morphogenetic protein 4 (BMP4) to serum-free ESC cultures can induce primitive streak-like mesodermal cells. In differentiated mouse ESCs, platelet-derived growth factor receptor-α (PDGFR-α) and E-cadherin (ECD) are useful markers to distinguish between paraxial mesodermal progenitor cells and undifferentiated and endodermal cells, respectively. Here, we demonstrate methods for BMP4-mediated induction of paraxial mesodermal progenitors using PDGFR-α and ECD as markers for purification and characterization. Serum-free monolayers of ESCs cultured with BMP4 could efficiently promote paraxial mesodermal differentiation akin to embryonic mesodermal development. BMP4 treatment alone induced paraxial mesodermal progenitors that could differentiate into osteochondrogenic cells in vitro and in vivo. Furthermore, early removal of BMP4 followed by lithium chloride (LiCl) promoted the differentiation to myogenic progenitor cells. These myogenic progenitors were able to differentiate further in vitro into mature skeletal muscle cells. Thus, we successfully induced the efficient bidirectional differentiation of mouse ESCs toward osteochondrogenic and myogenic cell types using chemically defined conditions.     

A novel serum-free monolayer culture for orderly hematopoietic differentiation of human pluripotent cells via mesodermal progenitors

Elucidating the in vitro differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells is important for understanding both normal and pathological hematopoietic development in vivo. For this purpose, a robust and simple hematopoietic differentiation system that can faithfully trace in vivo hematopoiesis is necessary. In this study, we established a novel serum-free monolayer culture that can trace the in vivo hematopoietic pathway from ES/iPS cells to functional definitive blood cells via mesodermal progenitors. Stepwise tuning of exogenous cytokine cocktails induced the hematopoietic mesodermal progenitors via primitive streak cells. These progenitors were then differentiated into various cell lineages depending on the hematopoietic cytokines present. Moreover, single cell deposition assay revealed that common bipotential hemoangiogenic progenitors were induced in our culture. Our system provides a new, robust, and simple method for investigating the mechanisms of mesodermal and hematopoietic differentiation.     

Compartment-dependent activities of Wnt3a/β-catenin signaling during vertebrate axial extension

Extension of the vertebrate body results from the concerted activity of many signals in the posterior embryonic end. Among them, Wnt3a has been shown to play relevant roles in the regulation of axial progenitor activity, mesoderm formation and somitogenesis. However, its impact on axial growth remains to be fully understood. Using a transgenic approach in the mouse, we found that the effect of Wnt3a signaling varies depending on the target tissue. High levels of Wnt3a in the epiblast prevented formation of neural tissues, but did not impair axial progenitors from producing different mesodermal lineages. These mesodermal tissues maintained a remarkable degree of organization, even within a severely malformed embryo. However, from the cells that failed to take a neural fate, only those that left the epithelial layer of the epiblast activated a mesodermal program. The remaining tissue accumulated as a folded epithelium that kept some epiblast-like characteristics. Together with previously published observations, our results suggest a dose-dependent role for Wnt3a in regulating the balance between renewal and selection of differentiation fates of axial progenitors in the epiblast. In the paraxial mesoderm, appropriate regulation of Wnt/β-catenin signaling was required not only for somitogenesis, but also for providing proper anterior–posterior polarity to the somites. Both processes seem to rely on mechanisms with different requirements for feedback modulation of Wnt/β-catenin signaling, once segmentation occurred in the presence of high levels of Wnt3a in the presomitic mesoderm, but not after permanent expression of a constitutively active form of β-catenin. Together, our findings suggest that Wnt3a/β-catenin signaling plays sequential roles during posterior extension, which are strongly dependent on the target tissue. This provides an additional example of how much the functional output of signaling systems depends on the competence of the responding cells.     

Deciphering the hierarchy of angiohematopoietic progenitors from human pluripotent stem cells

Identification of sequential progenitors leading to blood formation from pluripotent stem cells (PSCs) will be essential for understanding the molecular mechanisms of hematopoietic lineage specification and for development of technologies for in vitro production of hematopoietic stem cells (HSCs). It is well established that during development, blood and endothelial cells in the extraembryonic and embryonic compartments are formed in parallel from precursors with angiogenic and hematopoietic potentials. However, the identity and hierarchy of these precursors in human PSC (hPSC) cultures remain obscure. Using developmental stage-specific mesodermal and endothelial markers and functional assays, we recently identified discrete populations of angiohematopoietic progenitors from hPSCs, including mesodermal precursors and hemogenic endothelial cells with primitive and definitive hematopoietic potentials. In addition, we discovered a novel population of multipotent hematopoietic progenitors with an erythroid phenotype, which retain angiogenic potential. Here we introduce our recent findings and discuss their implication for defining putative HSC precursor and factors required for activation of self-renewal potential in hematopoietic cells emerging from endothelium.     

Tissue Differentiation in Mouse Teratocarcinomas in Lung Colonies from Ascitic Embryoid Bodies

Tissue differentiation in ascitic teratocarcinoma embryoid bodies (EBs) was investigated in the lung colony system of syngeneic 129/Sv and allogeneic Balb/c mice previously reported by us. In this report, tissues are classified as neural, epithelial and mesodermal tissues. Although multiple differentiation in these three groups of tissues became evident about a month following an injection of EBs, colonies containing only one type of tissue but not mesodermal were commonly found. There were few colonies containing only mesodermal or both neural and mesodermal tissues. This suggests that mesodermal tissue differentiation takes place, as in normal embryogenesis, through the formation of a mesodermal layer, which has not been found in vivo.     

The adult human brain harbors multipotent perivascular mesenchymal stem cells

Blood vessels and adjacent cells form perivascular stem cell niches in adult tissues. In this perivascular niche, a stem cell with mesenchymal characteristics was recently identified in some adult somatic tissues. These cells are pericytes that line the microvasculature, express mesenchymal markers and differentiate into mesodermal lineages but might even have the capacity to generate tissue-specific cell types. Here, we isolated, purified and characterized a previously unrecognized progenitor population from two different regions in the adult human brain, the ventricular wall and the neocortex. We show that these cells co-express markers for mesenchymal stem cells and pericytes in vivo and in vitro, but do not express glial, neuronal progenitor, hematopoietic, endothelial or microglial markers in their native state. Furthermore, we demonstrate at a clonal level that these progenitors have true multilineage potential towards both, the mesodermal and neuroectodermal phenotype. They can be epigenetically induced in vitro into adipocytes, chondroblasts and osteoblasts but also into glial cells and immature neurons. This progenitor population exhibits long-term proliferation, karyotype stability and retention of phenotype and multipotency following extensive propagation. Thus, we provide evidence that the vascular niche in the adult human brain harbors a novel progenitor with multilineage capacity that appears to represent mesenchymal stem cells and is different from any previously described human neural stem cell. Future studies will elucidate whether these cells may play a role for disease or may represent a reservoir that can be exploited in efforts to repair the diseased human brain.     

In Vitro Modeling of Paraxial Mesodermal Progenitors Derived from Induced Pluripotent Stem Cells

Induced pluripotent stem (iPS) cells are generated from adult somatic cells by transduction of defined factors. Given their unlimited proliferation and differentiation potential, iPS cells represent promising sources for cell therapy and tools for research and drug discovery. However, systems for the directional differentiation of iPS cells toward paraxial mesodermal lineages have not been reported. In the present study, we established a protocol for the differentiation of mouse iPS cells into paraxial mesodermal lineages in serum-free culture. The protocol was dependent on Activin signaling in addition to BMP and Wnt signaling which were previously shown to be effective for mouse ES cell differentiation. Independently of the cell origin, the number of transgenes, or the type of vectors used to generate iPS cells, the use of serum-free monolayer culture stimulated with a combination of BMP4, Activin A, and LiCl enabled preferential promotion of mouse iPS cells to a PDGFR-α⁺/Flk-1⁻ population, which represents a paraxial mesodermal lineage. The mouse iPS cell-derived paraxial mesodermal cells exhibited differentiation potential into osteogenic, chondrogenic, and myogenic cells both in vitro and in vivo and contributed to muscle regeneration. Moreover, purification of the PDGFR-α⁺/KDR⁻ population after differentiation allowed enrichment of human iPS cell populations with paraxial mesodermal characteristics. The resultant PDGFR-α⁺/KDR⁻ population derived from human iPS cells specifically exhibited osteogenic, chondrogenic, and myogenic differentiation potential in vitro, implying generation of paraxial mesodermal progenitors similar to mouse iPS cell-derived progenitors. These findings highlight the potential of protocols based on the serum-free, stepwise induction and purification of paraxial mesodermal cell lineages for use in stem cell therapies to treat diseased bone, cartilage, and muscle.     

Shield formation at the onset of zebrafish gastrulation

During vertebrate gastrulation, the three germ layers, ectoderm, mesoderm and endoderm are formed, and the resulting progenitor cells are brought into the positions from which they will later contribute more complex tissues and organs. A core element in this process is the internalization of mesodermal and endodermal progenitors at the onset of gastrulation. Although many of the molecules that induce mesendoderm have been identified, much less is known about the cellular mechanisms underlying mesendodermal cell internalization and germ layer formation. Here we show that at the onset of zebrafish gastrulation, mesendodermal progenitors in dorsal/axial regions of the germ ring internalize by single cell delamination. Once internalized, mesendodermal progenitors upregulate E-Cadherin (Cadherin 1) expression, become increasingly motile and eventually migrate along the overlying epiblast (ectodermal) cell layer towards the animal pole of the gastrula. When E-Cadherin function is compromised, mesendodermal progenitors still internalize, but, with gastrulation proceeding, fail to elongate and efficiently migrate along the epiblast, whereas epiblast cells themselves exhibit reduced radial cell intercalation movements. This indicates that cadherin-mediated cell-cell adhesion is needed within the forming shield for both epiblast cell intercalation, and mesendodermal progenitor cell elongation and migration during zebrafish gastrulation. Our data provide insight into the cellular mechanisms underlying mesendodermal progenitor cell internalization and subsequent migration during zebrafish gastrulation, and the role of cadherin-mediated cell-cell adhesion in these processes.     

Vessel-associated stem cells from skeletal muscle: From biology to future uses in cell therapy

Over the last years, the existence of different stem cells with myogenic potential has been widely investigated. Besides the classical skeletal muscle progenitors represented by satellite cells, numerous multipotent and embryologically unrelated progenitors with a potential role in muscle differentiation and repair have been identified. In order to conceive a therapeutic approach for degenerative muscle disorders, it is of primary importance to identify an ideal stem cell endowed with all the features for a possible use in vivo. Among all emerging populations, vessel-associated stem cells are a novel and promising class of multipotent progenitors of mesodermal origin and with high myogenic potential which seem to best fit all the requirements for a possible cell therapy. In vitro and in vivostudies have already tested the effectiveness and safety of vessel-associated stem cells in animal models. This leads to the concrete possibility in the future to start pilot human clinical trials, hopefully opening the way to a turning point in the treatment of genetic and acquired muscle disorders.     

Immature multipotent hemopoietic progenitors lacking long-term bone marrow-reconstituting activity in the aorta-gonad-mesonephros region of murine day 10 fetuses

Previous studies indicated that multipotent progenitors exist in early fetuses that do not contain long-term reconstituting (LTR) activity. However, it remained unclear whether these multipotent progenitors are committed to the hemopoietic lineage or are immature mesodermal cells or hemangioblasts. In this study, we have succeeded in enriching the multipotent progenitors that are capable of generating myeloid, T, and B cells in the LFA-1(-) subpopulation of TER-119(-)c-kit(+)CD45(+) cells from the aorta-gonad-mesonephros (AGM) region of day 10 fetuses. We found that these day 10 AGM LFA-1(-) cells do not show the LTR activity, whereas day 11 AGM LFA-1(-) cells do have such an activity. These results strongly suggest that multipotent progenitors lacking LTR activity emerge as CD45(+) hemopoietic progenitor cells in the AGM region on the 10th day of gestation, and such p-Multi mature into hemopoietic stem cells by acquiring LTR activity.     

Formation of mesodermal pattern by secondary inducing interactions

Summary A highly purified vegetalizing factor induces endoderm preferentially in amphibian gastrula ectoderm. After combination of this factor with less pure fractions, a high percentage of trunks and tails with notochord and somites are induced. The induction of these mesodermal tissues depends on secondary factors which may act on plasma membrane receptors of the target cells. The secondary factors are probably proteins as they are inactivated by trypsin or cellulose-bound proteinase K. They are not inactivated by thioglycolic acid.The implication of these findings for tissue determination and differentiation in normal development in relation to the anlageplan for endoderm and mesodermal tissues is discussed.     

Xenopus Dishevelled signaling regulates both neural and mesodermal convergent extension: parallel forces elongating the body axis

During amphibian development, non-canonical Wnt signals regulate the polarity of intercalating dorsal mesoderm cells during convergent extension. Cells of the overlying posterior neural ectoderm engage in similar morphogenetic cell movements. Important differences have been discerned in the cell behaviors associated with neural and mesodermal cell intercalation, raising the possibility that different mechanisms may control intercalations in these two tissues. In this report, targeted expression of mutants of Xenopus Dishevelled (Xdsh) to neural or mesodermal tissues elicited different defects that were consistent with inhibition of either neural or mesodermal convergent extension. Expression of mutant Xdsh also inhibited elongation of neural tissues in vitro in Keller sandwich explants and in vivo in neural plate grafts. Targeted expression of other Wnt signaling antagonists also inhibited neural convergent extension in whole embryos. In situ hybridization indicated that these defects were not due to changes in cell fate. Examination of embryonic phenotypes after inhibition of convergent extension in different tissues reveals a primary role for mesodermal convergent extension in axial elongation, and a role for neural convergent extension as an equalizing force to produce a straight axis. This study demonstrates that non-canonical Wnt signaling is a common mechanism controlling convergent extension in two very different tissues in the Xenopus embryo and may reflect a general conservation of control mechanisms in vertebrate convergent extension.     

Delayed differentiation in embryonic stem cells and mesodermal progenitors in the absence of CtBP2

Mammalian embryonic stem cells (ESCs) are characterized by an ability to self-renew and give rise to each of the three germ layers. ESCs are a pluripotential source of numerous primitive progenitors and committed lineages and can make stoichiometric decisions leading to either asymmetric or symmetric cell division. Several genes have been identified as essential for maintenance of self-renewal, but few non-lineage specific genes have been identified as essential for differentiation. We selected the chromatin factor Ctbp2 from microarray data for its enriched expression in stem cells, in comparison to committed progenitors. RNA interference (RNAi) was used to knockdown gene expression in mouse ESCs and the potential for transduced cells to self-renew and differentiate was assessed in ESC and mesodermal assays. Here, we demonstrate an important role for Ctbp2 in stem cell maintenance and regulation of differentiation using an in vitro system. The knockdown of Ctbp2 increases the prevalence of ESCs in culture, delays differentiation induced by LIF withdrawal, and introduces developmental changes in mesodermal differentiation. A model is presented for the importance of Ctbp2 in maintaining a balance in decisions to self-renewal and differentiate.