Lior Zangi

Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction

In a cell-free approach to regenerative therapeutics, transient application of paracrine factors in vivo could be used to alter the behavior and fate of progenitor cells to achieve sustained clinical benefits. Here we show that intramyocardial injection of synthetic modified RNA (modRNA) encoding human vascular endothelial growth factor-A (VEGF-A) results in the expansion and directed differentiation of endogenous heart progenitors in a mouse myocardial infarction model. VEGF-A modRNA markedly improved heart function and enhanced long-term survival of recipients. This improvement was in part due to mobilization of epicardial progenitor cells and redirection of their differentiation toward cardiovascular cell types. Direct in vivo comparison with DNA vectors and temporal control with VEGF inhibitors revealed the greatly increased efficacy of pulse-like delivery of VEGF-A. Our results suggest that modRNA is a versatile approach for expressing paracrine factors as cell fate switches to control progenitor cell fate and thereby enhance long-term organ repair.

Isolation of mesenchymal stem cells from G-CSF-mobilized human peripheral blood using fibrin microbeads.

Adult mesenchymal stem cells (MSC) that are able to differentiate into various mesenchymal cell types are typically isolated from bone marrow, but their significant presence in human peripheral blood (PB) is controversial. Fibrin microbeads (FMB) that bind matrix-dependent cells were used to isolate MSC from the mononuclear fraction of mobilized PB of adult healthy human donors treated with a granulocyte colony-stimulating factor. Isolation by plastic adherence resulted in a negligible number of MSC in all samples tested, whereas FMB-based isolation yielded spindle-shaped cell samples that could further expand on plastic or on FMB in eight out of the 11 samples. The yield of these cells at days 17–18 after the harvest was ∼0.5% of the initial cell number. The isolated cells were grown on plastic and characterized by FACS analysis and immunohistochemistry for specific markers. Following culturing and first passage, the FMB-isolated cells stained positive for mesenchymal stromal cell markers CD90 and CD105, expressed vimentin and fibronectin and were negative for hematopoietic markers CD45 and CD34. These cells could differentiate into osteoblasts, adipocytes and chondrocytes. This study indicates that FMB may have special advantage in isolating MSC from sources such as mobilized PB, where the number of such cells is scarce.

Isolation and Characterization of Nontubular Sca-1+Lin− Multipotent Stem/Progenitor Cells from Adult Mouse Kidney

Tissue engineering and cell therapy approaches aim to take advantage of the repopulating ability and plasticity of multipotent stem cells to regenerate lost or diseased tissue. Recently, stage-specific embryonic kidney progenitor tissue was used to regenerate nephrons. Through fluorescence-activated cell sorting, microarray analysis, in vitro differentiation assays, mixed lymphocyte reaction, and a model of ischemic kidney injury, this study sought to identify and characterize multipotent organ stem/progenitor cells in the adult kidney. Herein is reported the existence of nontubular cells that express stem cell antigen-1 (Sca-1). This population of small cells includes a CD45-negative fraction that lacks hematopoietic stem cell and lineage markers and resides in the renal interstitial space. In addition, these cells are enriched for beta1-integrin, are cytokeratin negative, and show minimal expression of surface markers that typically are found on bone marrow-derived mesenchymal stem cells. Global gene profiling reveals enrichment for many genes downstream of developmental signaling molecules and self-renewal pathways, such as TGF-beta/bone morphogenic protein, Wnt, or fibroblast growth factor, as well as for those that are involved in specification of mesodermal lineages (myocyte enhancer factor 2A, YY1-associated factor 2, and filamin-beta). In vitro, they are plastic adherent and slowly proliferating and result in inhibition of alloreactive CD8(+) T cells, indicative of an immune-privileged behavior. Furthermore, clonal-derived lines can be differentiated into myogenic, osteogenic, adipogenic, and neural lineages. Finally, when injected directly into the renal parenchyma, shortly after ischemic/reperfusion injury, renal Sca-1(+)Lin(-) cells, derived from ROSA26 reporter mice, adopt a tubular phenotype and potentially could contribute to kidney repair. These data define a unique phenotype for adult kidney-derived cells, which have potential as stem cells and may contribute to the regeneration of injured kidneys.

Direct imaging of immune rejection and memory induction by allogeneic mesenchymal stromal cells.

Although mesenchymal stromal cells (MSCs) exhibit marked immunoregulatory activity through multiple mechanisms, their potential to completely evade rejection upon transplantation into allogeneic recipients is controversial. To directly address this controversy, the survival of luciferase‐labeled MSCs (Luc+ MSCs) was evaluated by imaging in allogeneic recipients. This analysis showed that although MSCs exhibited longer survival compared to fibroblasts (Fib), their survival was significantly shorter compared to that exhibited in syngeneic or in immune‐deficient Balb‐Nude or non‐obese diabetic severe combined immunodeficiency (NOD‐SCID) recipients. Graft rejection in re‐challenge experiments infusing Luc+ Fib into mice, which had previously rejected Luc+ MSCs, indicated potential induction of immune memory by the MSCs. This was further analyzed in T‐cell antigen receptor (TCR) transgeneic mice in which either CD4 TEA mice or CD8 T cells (2C mice) bear a TCR transgene against a specific MHC I or MHC II, respectively. Thus, following a re‐challenge with MSCs expressing the cognate MHC haplotype, an enhanced percentage of 2C CD8+ or TEA CD4+ T cells exhibited a memory phenotype (CD122+, CD44+, and CD62Llow). Collectively, these results demonstrate that MSCs are not intrinsically immune‐privileged, and under allogeneic settings, these cells induce rejection, which is followed by an immune memory. Considering that the use of allogeneic or even a third party (“off the shelf”) MSCs is commonly advocated for a variety of clinical applications, our results strongly suggest that long‐term survival of allogeneic MSCs likely represents a major challenge. STEM CELLS 2009;27:2865–2874

A HCN4+ cardiomyogenic progenitor derived from the first heart field and human pluripotent stem cells

Most of the mammalian heart is formed from mesodermal progenitors in the first and second heart fields (FHF and SHF), whereby the FHF gives rise to the left ventricle and parts of the atria and the SHF to the right ventricle, outflow tract and parts of the atria. Whereas SHF progenitors have been characterized in detail, using specific molecular markers, comprehensive studies on the FHF have been hampered by the lack of exclusive markers. Here, we present Hcn4 (hyperpolarization-activated cyclic nucleotide-gated channel 4) as an FHF marker. Lineage-traced Hcn4+/FHF cells delineate FHF-derived structures in the heart and primarily contribute to cardiomyogenic cell lineages, thereby identifying an early cardiomyogenic progenitor pool. As a surface marker, HCN4 also allowed the isolation of cardiomyogenic Hcn4+/FHF progenitors from human embryonic stem cells. We conclude that a primary purpose of the FHF is to generate cardiac muscle and support the contractile activity of the primitive heart tube, whereas SHF-derived progenitors contribute to heart cell lineage diversification.

How to make a cardiomyocyte

During development, cardiogenesis is orchestrated by a family of heart progenitors that build distinct regions of the heart. Each region contains diverse cell types that assemble to form the complex structures of the individual cardiac compartments. Cardiomyocytes are the main cell type found in the heart and ensure contraction of the chambers and efficient blood flow throughout the body. Injury to the cardiac muscle often leads to heart failure due to the loss of a large number of cardiomyocytes and its limited intrinsic capacity to regenerate the damaged tissue, making it one of the leading causes of morbidity and mortality worldwide. In this Primer we discuss how insights into the molecular and cellular framework underlying cardiac development can be used to guide the in vitro specification of cardiomyocytes, whether by directed differentiation of pluripotent stem cells or via direct lineage conversion. Additional strategies to generate cardiomyocytes in situ, such as reactivation of endogenous cardiac progenitors and induction of cardiomyocyte proliferation, will also be discussed.

Driving vascular endothelial cell fate of human multipotent Isl1+ heart progenitors with VEGF modified mRNA

Distinct families of multipotent heart progenitors play a central role in the generation of diverse cardiac, smooth muscle and endothelial cell lineages during mammalian cardiogenesis. The identification of precise paracrine signals that drive the cell-fate decision of these multipotent progenitors, and the development of novel approaches to deliver these signals in vivo, are critical steps towards unlocking their regenerative therapeutic potential. Herein, we have identified a family of human cardiac endothelial intermediates located in outflow tract of the early human fetal hearts (OFT-ECs), characterized by coexpression of Isl1 and CD144/vWF. By comparing angiocrine factors expressed by the human OFT-ECs and non-cardiac ECs, vascular endothelial growth factor (VEGF)-A was identified as the most abundantly expressed factor, and clonal assays documented its ability to drive endothelial specification of human embryonic stem cell (ESC)-derived Isl1+ progenitors in a VEGF receptor-dependent manner. Human Isl1-ECs (endothelial cells differentiated from hESC-derived ISL1+ progenitors) resemble OFT-ECs in terms of expression of the cardiac endothelial progenitor- and endocardial cell-specific genes, confirming their organ specificity. To determine whether VEGF-A might serve as an in vivo cell-fate switch for human ESC-derived Isl1-ECs, we established a novel approach using chemically modified mRNA as a platform for transient, yet highly efficient expression of paracrine factors in cardiovascular progenitors. Overexpression of VEGF-A promotes not only the endothelial specification but also engraftment, proliferation and survival (reduced apoptosis) of the human Isl1+ progenitors in vivo. The large-scale derivation of cardiac-specific human Isl1-ECs from human pluripotent stem cells, coupled with the ability to drive endothelial specification, engraftment, and survival following transplantation, suggest a novel strategy for vascular regeneration in the heart.

Reconstruction of Cell Lineage Trees in Mice

The cell lineage tree of a multicellular organism represents its history of cell divisions from the very first cell, the zygote. A new method for high-resolution reconstruction of parts of such cell lineage trees was recently developed based on phylogenetic analysis of somatic mutations accumulated during normal development of an organism. In this study we apply this method in mice to reconstruct the lineage trees of distinct cell types. We address for the first time basic questions in developmental biology of higher organisms, namely what is the correlation between the lineage relation among cells and their (1) function, (2) physical proximity and (3) anatomical proximity. We analyzed B-cells, kidney-, mesenchymal- and hematopoietic-stem cells, as well as satellite cells, which are adult skeletal muscle stem cells isolated from their niche on the muscle fibers (myofibers) from various skeletal muscles. Our results demonstrate that all analyzed cell types are intermingled in the lineage tree, indicating that none of these cell types are single exclusive clones. We also show a significant correlation between the physical proximity of satellite cells within muscles and their lineage. Furthermore, we show that satellite cells obtained from a single myofiber are significantly clustered in the lineage tree, reflecting their common developmental origin. Lineage analysis based on somatic mutations enables performing high resolution reconstruction of lineage trees in mice and humans, which can provide fundamental insights to many aspects of their development and tissue maintenance.

Estimating cell depth from somatic mutations.

The depth of a cell of a multicellular organism is the number of cell divisions it underwent since the zygote, and knowing this basic cell property would help address fundamental problems in several areas of biology. At present, the depths of the vast majority of human and mouse cell types are unknown. Here, we show a method for estimating the depth of a cell by analyzing somatic mutations in its microsatellites, and provide to our knowledge for the first time reliable depth estimates for several cells types in mice. According to our estimates, the average depth of oocytes is 29, consistent with previous estimates. The average depth of B cells ranges from 34 to 79, linearly related to the mouse age, suggesting a rate of one cell division per day. In contrast, various types of adult stem cells underwent on average fewer cell divisions, supporting the notion that adult stem cells are relatively quiescent. Our method for depth estimation opens a window for revealing tissue turnover rates in animals, including humans, which has important implications for our knowledge of the body under physiological and pathological conditions.

High-Yield Isolation, Expansion, and Differentiation of Murine Bone Marrow-Derived Mesenchymal Stem Cells Using Fibrin Microbeads (FMB)

Transplantation of adult mesenchymal stem cells (MSCs) could provide a basis for tissue regeneration. MSCs are typically isolated from bone marrow (BM) based on their preferential adherence to plastic, although with low efficiency in terms of yield and purity. Extensive expansion is needed to reach a significant number of MSCs for any application. Fibrin microbeads (FMB) were designed to attach mesenchymal cells and to provide a matrix for their expansion. The current study was aimed at isolating a high yield of purified BM-derived mouse MSCs based on their preferential adherence and proliferation on FMB in suspension cultures. MSCs could be downloaded to plastics or further expanded on FMB. The yield of MSCs obtained by the FMB isolation technique was about one order of magnitude higher than that achieved by plastic adherence, suggesting that these cells are more abundant than previously reported. FMB-isolated cells were classified as MSCs by their fibroblastic morphology, self-renewal ability, and expression profile of their surface antigens, as examined by flow cytometry and immunostaining. In cell culture, the isolated MSCs could be induced to differentiate into three different mesodermal lineages, as demonstrated by histochemical stains and by RT-PCR analyses of tissue-specific genes. MSCs were also able to differentiate into osteocytes while still cultured on FMB. Our results suggest that FMB might serve as an efficient platform for the isolation, expansion, and differentiation of mouse BM-derived MSCs to be subsequently implanted for tissue regeneration.