Transplantation of human embryonic stem cell-derived alveolar epithelial type II cells abrogates acute lung injury in mice.

Respiratory diseases are a major cause of mortality and morbidity worldwide. Current treatments offer no prospect of cure or disease reversal. Transplantation of pulmonary progenitor cells derived from human embryonic stem cells (hESCs) may provide a novel approach to regenerate endogenous lung cells destroyed by injury and disease. Here, we examine the therapeutic potential of alveolar type II epithelial cells derived from hESCs (hES-ATIICs) in a mouse model of acute lung injury. When transplanted into lungs of mice subjected to bleomycin (BLM)-induced acute lung injury, hES-ATIICs behaved as normal primary ATIICs, differentiating into cells expressing phenotypic markers of alveolar type I epithelial cells. Without experiencing tumorigenic side effects, lung injury was abrogated in mice transplanted with hES-ATIICs, demonstrated by recovery of body weight and arterial blood oxygen saturation, decreased collagen deposition, and increased survival. Therefore, transplantation of hES-ATIICs shows promise as an effective therapeutic to treat acute lung injury.

Astrocytes from the contused spinal cord inhibit oligodendrocyte differentiation of adult oligodendrocyte precursor cells by increasing the expression of bone morphogenetic proteins.

Promotion of remyelination is an important therapeutic strategy to facilitate functional recovery after traumatic spinal cord injury (SCI). Transplantation of neural stem cells (NSCs) or oligodendrocyte precursor cells (OPCs) has been used to enhance remyelination after SCI. However, the microenvironment in the injured spinal cord is inhibitory for oligodendrocyte (OL) differentiation of NSCs or OPCs. Identifying the signaling pathways that inhibit OL differentiation in the injured spinal cord could lead to new therapeutic strategies to enhance remyelination and functional recovery after SCI. In the present study, we show that reactive astrocytes from the injured rat spinal cord or their conditioned media inhibit OL differentiation of adult OPCs with concurrent promotion of astrocyte differentiation. The expression of bone morphogenetic proteins (BMP) is dramatically increased in the reactive astrocytes and their conditioned media. Importantly, blocking BMP activity by BMP receptor antagonist, noggin, reverse the effects of active astrocytes on OPC differentiation by increasing the differentiation of OL from OPCs while decreasing the generation of astrocytes. These data indicate that the upregulated bone morphogenetic proteins in the reactive astrocytes are major factors to inhibit OL differentiation of OPCs and to promote its astrocyte differentiation. These data suggest that manipulation of BMP signaling in the endogenous or grafted NSCs or OPCs may be a useful therapeutic strategy to increase their OL differentiation and remyelination and enhance functional recovery after SCI.

BMP-SIGNALING REGULATES A COMMON TRANSCRIPTIONAL PROGRAM TO CONTROL FACIAL FORM AND SKELETAL MORPHOGENESIS

Much of the craniofacial skeleton, such as the skull vault, mandible and midface, develops through direct, intramembranous ossification of the cranial neural crest (CNC) derived progenitor cells. Bmp-signaling plays critical roles in normal craniofacial development, and Bmp4 deficiency results in craniofacial abnormalities, such as cleft lip and palate. We performed an in depth analysis of Bmp4, a critical regulator of development, disease, and evolution, in the CNC. Conditional Bmp4 overexpression, using a tetracycline regulated Bmp4 gain of function allele, resulted in facial form changes that were most dramatic after an E10.5 Bmp4 induction. Expression profiling uncovered a signature of Bmp4 induced genes (BIG) composed predominantly of transcriptional regulators controlling self-renewal, osteoblast differentiation, and negative Bmp autoregulation. The complimentary experiment, CNC inactivation of Bmp2, Bmp4, and Bmp7, resulted in complete or partial loss of multiple CNC derived skeletal elements revealing a critical requirement for Bmp-signaling in membranous bone and cartilage development. Importantly, the BIG signature was reduced in Bmp loss of function mutants indicating similar Bmp-regulated target genes underlying facial form modulation and normal skeletal morphogenesis. Chromatin immunoprecipitation (ChIP) revealed a subset of the BIG signature, including Satb2, Smad6, Hand1, Gadd45g and Gata3 that was bound by Smad1/5 in the developing mandible revealing direct, Smad-mediated regulation. These data indicate that Bmp-signaling regulates craniofacial skeletal development and facial form by balancing self-renewal and differentiation pathways in CNC progenitors.

Maintenance of phosphatidylserine asymmetry in erythroid cells during differentiation, aging, and its significance for macrophage recognition

Phosphatidylserine (PS) is distributed almost entirely in the inner leaflet of the erythrocyte membrane bilayer, and appears to be maintained by a 32 kDa integral membrane protein (PS translocase). The expression of PS on the outer leaflet may serve as a recognition signal for macrophages, since insertion of PS into erythrocytes enhances their adherence to macrophages and clearance from the circulation. Therefore I have hypothesized that erythroid cells display PS on their outer leaflet early in differentiation and upon aging. Analysis of murine erythroleukemia cells (MELC, undifferentiated erythroid progenitor cells) showed high levels of PS on the outer leaflet that decreased during differentiation, correlating with the pattern of macrophage adherence. The activity of the PS translocase during differentiation appears to be unchanged although the equilibrium distribution of PS differs. This difference may be due to qualitative changes in the PS translocase. $\sp{125}$I-Bolton/Hunter-labeled-pyridyldithioethylamine ($\sp{125}$I-B/H-PDA), a radiolabeled probe for the PS translocase, labeled a 32 kDa protein in mature erythrocytes whereas in MELC a 45 kDa protein as well as a 32 kDa protein was identified. The abundance of the 45 kDa protein in relation to the 32 kDa protein declined during differentiation, possibly indicating this protein was a precursor of the 32 kDa protein. Analysis of the 45 kDa protein by N-glycosidase F and endoproteinase cleavage suggested this protein was not a glycosylated form of the 32 kDa protein but appeared to share some structural homology. Aged murine erythrocytes had elevated levels of PS on their outer leaflet, as well as decreased PS translocase activity. $\sp{125}$I-B/H-PDA labeled a 32 kDa protein in both normal and aged erythrocytes. However, the latter cells also contained a 28 kDa protein. Experimental evidence suggests that the appearance of the 28 kDa protein may be due to increased oxidation of aged erythrocytes. Examination of PS distribution showed that the levels of PS on the outer leaflet were elevated early in differentiation, decreased during the mature state, and returned to high levels as the erythrocyte aged. In conclusion,the levels of outer leaflet PS correlated with the differentiation status and macrophage recognition of erythroid cells. ^

Rescue from apoptosis in early (CD34 selected) versus late (CD34 unselected) human hematopoietic cells by VLA-4 and VCAM-1 dependent adhesion to bone marrow stromal cells

The interaction of hematopoietic precursor cell with bone marrow stromal cells is assumed to be important to the survival of hematopoietic precursor cells during hematopoietic cell long-term culture. Early hematopoietic stem cells are preferentially found within the stromal adherent cell fraction in primary long-term bone marrow cultures. The purpose of this dissertation was to understand the molecular mechanisms that govern these interactions for the regulation of survival and proliferation of early versus late hematopoietic cells.^ Monoclonal antibodies to the VLA-4 recognize the alpha4 beta1 integrin receptor on human hematopoietic cells. This monoclonal antibody blocks the adhesion between early hematopoietic progenitor cells (CD34 selected cells) and stromal cells when added to cultures of these cells. Addition of the VLA-4 monoclonal antibody to cultures of stromal cells and CD34 selected cells was shown to induce apoptosis of CD34 selected cells in these CD34 selected cell/stromal cell cocultures, as measured by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling method. In contrast to these experiments with early hematopoietic progenitor cells (CD34+), the level of adhesion between more differentiated cells (unfractionated hematopoietic cells) and stromal cells was not significantly altered by addition of the anti-VLA-4 monoclonal antibody. Similarly, the level of apoptosis of unfractionated hematopoietic cells was not significantly increased by the addition of anti-VLA-4 monoclonal antibody to cultures of the latter cells with stromal cells. The binding of the unfractionated cells is less than that of the CD34 selected. Since there is no difference between the alpha4 beta1 integrin expression level of the early and late myeloid cells, there may be a difference in the functional state of the integrin between the early and late myeloid cells. We also show that CD34+ selected precursor cells proliferate at a higher rate when these cells are plated on recombinant VCAM-1 molecules. These data indicate that the alpha4beta1 integrin receptor (VLA-4) plays a central role in the apoptosis rescue function which results from the anchorage-dependent growth of the CD34 selected early hematopoietic cells on stromal cells. The data suggest that these apoptosis rescue pathways have less significance as the cells mature and become anchorage-independent in their growth. These data should assist in the design of systems for the ex vivo proliferation and transduction of early hematopoietic cells for genetic therapy. ^

Regulation of hematopoietic and leukemic stem cells by the immune system.

Hematopoietic stem cells (HSCs) are rare, multipotent cells that generate via progenitor and precursor cells of all blood lineages. Similar to normal hematopoiesis, leukemia is also hierarchically organized and a subpopulation of leukemic cells, the leukemic stem cells (LSCs), is responsible for disease initiation and maintenance and gives rise to more differentiated malignant cells. Although genetically abnormal, LSCs share many characteristics with normal HSCs, including quiescence, multipotency and self-renewal. Normal HSCs reside in a specialized microenvironment in the bone marrow (BM), the so-called HSC niche that crucially regulates HSC survival and function. Many cell types including osteoblastic, perivascular, endothelial and mesenchymal cells contribute to the HSC niche. In addition, the BM functions as primary and secondary lymphoid organ and hosts various mature immune cell types, including T and B cells, dendritic cells and macrophages that contribute to the HSC niche. Signals derived from the HSC niche are necessary to regulate demand-adapted responses of HSCs and progenitor cells after BM stress or during infection. LSCs occupy similar niches and depend on signals from the BM microenvironment. However, in addition to the cell types that constitute the HSC niche during homeostasis, in leukemia the BM is infiltrated by activated leukemia-specific immune cells. Leukemic cells express different antigens that are able to activate CD4(+) and CD8(+) T cells. It is well documented that activated T cells can contribute to the control of leukemic cells and it was hoped that these cells may be able to target and eliminate the therapy-resistant LSCs. However, the actual interaction of leukemia-specific T cells with LSCs remains ill-defined. Paradoxically, many immune mechanisms that evolved to activate emergency hematopoiesis during infection may actually contribute to the expansion and differentiation of LSCs, promoting leukemia progression. In this review, we summarize mechanisms by which the immune system regulates HSCs and LSCs.

Cytotoxic CD8+ T cells stimulate hematopoietic progenitors by promoting cytokine release from bone marrow mesenchymal stromal cells.

Cytotoxic CD8(+) T cells (CTLs) play a major role in host defense against intracellular pathogens, but a complete clearance of pathogens and return to homeostasis requires the regulated interplay of the innate and acquired immune systems. Here, we show that interferon γ (IFNγ) secreted by effector CTLs stimulates hematopoiesis at the level of early multipotent hematopoietic progenitor cells and induces myeloid differentiation. IFNγ did not primarily affect hematopoietic stem or progenitor cells directly. Instead, it promoted the release of hematopoietic cytokines, including interleukin 6 from bone marrow mesenchymal stromal cells (MSCs) in the hematopoietic stem cell niche, which in turn reduced the expression of the transcription factors Runx-1 and Cebpα in early hematopoietic progenitor cells and increased myeloid differentiation. Therefore, our study indicates that, during an acute viral infection, CTLs indirectly modulate early multipotent hematopoietic progenitors via MSCs in order to trigger the temporary activation of emergency myelopoiesis and promote clearance of the infection.

Lymph node stromal cells negatively regulate antigen-specific CD4+ T cell responses

Lymph node (LN) stromal cells (LNSCs) form the functional structure of LNs and play an important role in lymphocyte survival and the maintenance of immune tolerance. Despite their broad spectrum of function, little is known about LNSC responses during microbial infection. In this study, we demonstrate that LNSC subsets display distinct kinetics following vaccinia virus infection. In particular, compared with the expansion of other LNSC subsets and the total LN cell population, the expansion of fibroblastic reticular cells (FRCs) was delayed and sustained by noncirculating progenitor cells. Notably, newly generated FRCs were preferentially located in perivascular areas. Viral clearance in reactive LNs preceded the onset of FRC expansion, raising the possibility that viral infection in LNs may have a negative impact on the differentiation of FRCs. We also found that MHC class II expression was upregulated in all LNSC subsets until day 10 postinfection. Genetic ablation of radioresistant stromal cell-mediated Ag presentation resulted in slower contraction of Ag-specific CD4(+) T cells. We propose that activated LNSCs acquire enhanced Ag-presentation capacity, serving as an extrinsic brake system for CD4(+) T cell responses. Disrupted function and homeostasis of LNSCs may contribute to immune deregulation in the context of chronic viral infection, autoimmunity, and graft-versus-host disease.

Mechanical Loading Promoted Discogenic Differentiation of Human Mesenchymal Stem Cells Incorporated in 3D-PEG Scaffolds with RhGDF5 and RGD

Hydrogels have been described as ideal scaffolds for cells of 3D tissue constructs and hold strong promises with respect to in vitro 3D-cell-culture, where cells are isolated from native extracellular matrix (ECM). Synthesized polyethyleneglycol (PEG) hydrogels are appealing with regard to potential for cell therapy or as vehicles for drug delivery or even to regenerate tissue with similar hydrogel-like properties such as the nucleus pulposus of the intervertebral disc (IVD). Here, we tested whether incorporation of RGD motive would hinder discogenic differentiation of primary bone marrow-derived human mesenchymal stem cells (hMSCs) but favor proliferation of undifferentiated hMSCs. HMSCs were embedded in +RGD containing or without RGD PEG hydrogel and pre-conditioned with or without growth and differentiation factor-5 (rhGDF-5) for 13 days. Afterwards, all hMSCs-PEG gels were subsequently cyclically loaded (15% strain, 1Hz) for 5 consecutive days in a bioreactor to generate an IVD-like phenotype. Higher metabolic activity (resazurin assay) was found in groups with rhGDF5 in both gel types with and without RGD. Cell viability and morphology measured by confocal laser microscopy and DNA content showed decreased values (~60%) after 18 days of culture. Real-time RT-PCR of an array of 15 key genes suspected to be distinctive for IVD cells revealed moderate response to rhGDF5 and mechanical loading as also shown by histology staining. Preconditioning and mechanical loading showed relatively moderate responses revealed from both RT-PCR and histology although hMSCs were demonstrated to be potent to differentiate into chondrocyte-progenitor cells in micro- mass and 3D alginate bead culture.

Bovine coccygeal intervertebral discs contain multipotent Tie2+ cells which can differentiate into osteogenic and adipogenic lineages

Question: The intervertebral disc (IVD) has a limited regenerative potential and low back pain represents a leading cause of disability [1]. IVD repair strategies require an appropriate cell source that is able to regenerate the damaged tissue such as progenitor stem cells. Recently, progenitor cells that are positive for the angiopoietin re- ceptor (Tie2) in the nucleus pulposus were identified [2]. Here we isolated primary cells from bovine IVD and sorted bovine nucleus pulposus progenitor cells (NPPC) for the marker Tie2. Furthermorewe tested whether Tie2 expressing cells can differentiate into os- teogenic and adipogenic lineages in vitro. Methods: NP cells were obtained from 1 year old bovine tails by sequential digestion with pronase for 1 h and collagenase over- night. Sorted Tie2- and Tie2+ cells were cultured in osteogenic and adipogenic medium for 3 weeks. The formed cell layers from both subpopulations were stained for calcium deposition and fat droplets. Colony forming units were prepared for both cell sus- pensions in methylcellulose-based medium and formed colonies ([10 cells) were analyzed macroscopically after 8 days. Results: After 3 weeks of culture, sorted Tie2+ cells were able to differentiate into osteocytes and adipocytes as characterized by cal- cium deposition and fat droplet formation. By contrast, Tie2- cells generated a weak staining for calcium and no fat droplets were ob- tained (Fig. 1). Sorted Tie2- and Tie2+ subpopulations of cells both formed colonies, however with different morphologies. The colonies formed from Tie2+ cells were spheroid in shape whereas those from Tie2- cells were spread and fibroblastic. Conclusion: Our data showed that Tie2+ cells of the nucleus pul- posus cells are progenitor-like cells that are able to differentiate into osteogenic and adipogenic lineages. Sorting of NPPC for Tie2 may represent a promising strategy with the potential to be used in the clinics for treatment of intervertebral disc damage. References 1. Freemont AJ (2009) The cellular pathobiology of the degenerate intervertebral disc and discogenic back pain. Rheumatology (Oxford) 48:5–10 2. Sakai D, Nakamura Y, Nakai T et al (2012) Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc. Nat Commun 3:1264 Acknowledgments: This project was funded by two projects of the Swiss National Science Foundation grant number #IZK0Z3_154384 and #310030_153411.

Low DICER1 expression is associated with attenuated neutrophil differentiation and autophagy of NB4 APL cells

Successful myeloid differentiation depends on the expression of a series of miRNAs. Thus, it is hardly surprising that miRNAs are globally repressed in AML, a disease mainly characterized by a block in cellular myeloid differentiation. Studies investigating the mechanisms for low miRNA expression in AML has mostly focused on altered transcriptional regulation or deletions, whereas defective miRNA processing has received less attention. In this study, we report that the expression of the key miRNA processing enzyme DICER1 is down-regulated in primary AML patient samples and healthy CD34(+) progenitor cells as compared with granulocytes. In line with these findings, Dicer1 expression was induced significantly in AML cell lines upon neutrophil differentiation. The knocking down of DICER1 in AML cells significantly attenuated neutrophil differentiation, which was paralleled by decreased expression of miRNAs involved in this process. Moreover, we found that inhibiting DICER1 attenuated the activation of autophagy, a cellular recycling process that is needed for proper neutrophil differentiation of AML cells. Our results clearly indicate that DICER1 plays a novel role in neutrophil differentiation as well as in myeloid autophagy of AML cells.

Tyrosine kinase inhibitor-induced CD70 expression mediates drug resistance in leukemia stem cells by activating Wnt signaling.

In chronic myelogenous leukemia (CML), oncogenic BCR-ABL1 activates the Wnt pathway, which is fundamental for leukemia stem cell (LSC) maintenance. Tyrosine kinase inhibitor (TKI) treatment reduces Wnt signaling in LSCs and often results in molecular remission of CML; however, LSCs persist long term despite BCR-ABL1 inhibition, ultimately causing disease relapse. We demonstrate that TKIs induce the expression of the tumor necrosis factor (TNF) family ligand CD70 in LSCs by down-regulating microRNA-29, resulting in reduced CD70 promoter DNA methylation and up-regulation of the transcription factor specificity protein 1. The resulting increase in CD70 triggered CD27 signaling and compensatory Wnt pathway activation. Combining TKIs with CD70 blockade effectively eliminated human CD34(+) CML stem/progenitor cells in xenografts and LSCs in a murine CML model. Therefore, targeting TKI-induced expression of CD70 and compensatory Wnt signaling resulting from the CD70/CD27 interaction is a promising approach to overcoming treatment resistance in CML LSCs.

[Lineage tracing of epicardial cells during development and regeneration].

Tracing the history of individual cells during embryonic morphogenesis in a structure as complex as the cardiovascular system is one of the major challenges of developmental biology. It involves determining the relationships between the various lineages of cells forming an organ at different stages, describing the topological rearrangements tissues undergo during morphogenesis, and characterizing the interactions between cells in different structures. However, despite the great expectations raised in the field of regenerative medicine, only limited progress has been made in using regenerative therapy to repair the cardiovascular system. Recent research has highlighted the role of the epicardium during cardiac regeneration, but it is still unclear whether it is important for molecular signaling or acts as a source of progenitor cells during this process. Consequently, increasing knowledge about the origin, diversification and potential of epicardial cells during development and homeostasis and under pathological conditions is of fundamental importance both for basic research and for the development of effective cellular therapies. The aims of this article were to provide a general overview of the classical techniques used for tracing cell lineages, including their potential and limitations, and to describe novel techniques for studying the origin and differentiation of the epicardium and its role in cardiac regeneration.

Extracellular Iron is a Modulator of the Differentiation of Osteoclast Lineage Cells.

Osteoclasts originate from the hematopoietic stem cell and share a differentiation pathway with the cells of the monocyte/macrophage lineages. Development and activation of osteoclasts, and as a consequence regulation of bone resorption, depend on two growth factors: macrophage colony-stimulating factor and receptor activator of NF-κB ligand. Furthermore, cell development and activity are modulated by a microenvironment composed of cytokines and growth factors and of the extracellular matrix. Membrane transporters are a means for cells to interact with their environment. Within this study, the expression of proteins regulating cellular iron homeostasis in osteoclast-like cells grown from bone marrow-derived progenitors was compared to the expression of this set of proteins by monocyte/macrophage lineage cells. In differentiating osteoclasts, levels of transcripts encoding transferrin receptor 1 and divalent metal transporter 1 (Slc11A2) were increased, while levels of transcripts encoding ferroportin (Slc40A1) and natural resistance-associated macrophage protein 1 (Slc11A1) were decreased. Supplementation of the culture media with exogenous iron led to an increase in the proliferation of osteoclast progenitor cells and to the expression of a macrophage-like phenotype, while the development of osteoclasts was reduced. Upon transfer of mature OC onto a CaP substrate, iron depletion of the medium with the Fe(3+)-chelator Deferoxamine Mesylate decreased CaP dissolution by ~30 %, which could be restored by addition of exogenous iron. During the 24 h of the assay, no effects were observed on total TRAP activity. The data demonstrate transcriptional regulation of the components of cellular iron transporters during OC development and suggests that iron homeostasis may contribute to fine-tuning of the RANKL-induced OC development.

On a mathematical model of tumor growth based on cancer stem cells

We consider a simple mathematical model of tumor growth based on cancer stem cells. The model consists of four hyperbolic equations of first order to describe the evolution of different subpopulations of cells: cancer stem cells, progenitor cells, differentiated cells and dead cells. A fifth equation is introduced to model the evolution of the moving boundary. The system includes non-local terms of integral type in the coefficients. Under some restrictions in the parameters we show that there exists a unique homogeneous steady state which is stable.