Self-renewing Progenitor Population Research Articles

Derivation of Multipotent Neural Progenitors from Human Embryonic Stem Cells for Cell Therapy and Biomedical Applications.

Long-term neuroepithelial-like stem cells (lt-NES) derived from human embryonic stem cells are a stable self-renewing progenitor population with high neurogenic potential and phenotypic plasticity. Lt-NES are amenable to regional patterning toward neurons and glia subtypes and thus represent a valuable source of cells for many biomedical applications. For use in regenerative medicine and cell therapy, lt-NES and their progeny require derivation with high-quality culture conditions suitable for clinical use. In this chapter, we describe a robust method to derive multipotent and expandable lt-NES based on good manufacturing practice and cell therapy-grade reagents. We further describe fully defined protocols to terminally differentiate lt-NES toward GABA-ergic, dopaminergic, and motor neurons.

CD4+ T cell help is required for the formation of a cytolytic CD8+ T cell subset that protects against chronic viral infection and cancer

Abstract CD8 T cells responding to chronic viral infection can be classified into two major subsets, with a TCF-1+ subset serving as a self-renewing progenitor population that can give rise to a more terminally exhausted PD-1hi subset. However, whether additional heterogeneity exists amongst virus-specific CD8 T cells remains unclear. Here, using single cell RNA-sequencing and flow cytometric approaches, we demonstrate that in addition to the progenitor and exhausted subsets, CD8 T cells responding to persistent LCMV infection also encompass a previously unrecognized T cell subset that exhibits potent cytolytic activity in vitro, and is required for viral control in vivo. This cytotoxic subset is characterized by high expression of killer cell lectin-like receptors, the chemokine receptor CX3CR1, and the transcription factors T-bet and Zeb2. Notably, we further demonstrate a critical role for CD4 help via IL-21 production in facilitating the differentiation of this cytotoxic subset, as depletion of CD4 T cells or deletion of IL-21R signaling in P14 cells (which express a transgenic TCR specific for GP33–41) wholly abrogated the development of CX3CR1+CD8 T cells. Moreover, PD-L1 blockade, an immunotherapy known to partially reverse T cell exhaustion, failed to rescue this differentiation defect of “un-helped” CD8 T cells. Lastly, using a B16 melanoma model, we found that provision of IL-21+CD4 T cells was sufficient to induce over a 2-fold increase in the development of CX3CR1+CD8 TILs, which was associated with enhanced control over tumor growth. Collectively, our findings demonstrate a critical role for CD4 help in the generation of protective CX3CR1+CD8 T cells, and that this differentiation checkpoint may be exploited for immunotherapy.

FXR1 expression domain in Wilms tumor

Background/PurposeWilms tumor (WT) is the most common childhood kidney cancer globally. Our prior unbiased proteomic screen of WT disparities revealed increased expression of Fragile X-Related 1 (FXR1) in Kenyan specimens where survival is dismal. FXR1 is an RNA-binding protein that associates with poor outcomes in multiple adult cancers. The aim of this study therefore was to validate and characterize the FXR1 expression domain in WT. MethodsQuantitative FXR1 gene expression was compared between WT, adjacent, adult, and fetal kidney specimens. The cellular and subcellular expression domain of FXR1 was characterized across these tissues using immunoperoxidase staining. RNA-sequencing of FXR1 was performed from WT and other pediatric malignancies to examine its broader target potential. ResultsFXR1 was detected in all clinical WT specimens evaluated (n = 82), and as a result appeared independent of demographic, histology, or adverse event. Specific cytosolic staining was strongest in blastema, intermediate and variable in epithelia, and weakest in stroma. When present, areas of skeletal muscle differentiation stained strongly for FXR1. qPCR revealed increased FXR1 expression in WT compared to adult and adjacent kidney (p < 0.0002) but was similar to fetal kidney (p = 0.648). RNA-sequencing revealed expression of FXR1 in multiple pediatric tumors, greatest in rhabdomyosarcoma and WT. ConclusionsFXR1 was expressed consistently across this broad sampling of WT and most robustly in the primitive blastema. Notably, FXR1 labeled a specific self-renewing progenitor population of the fetal kidney.

Identification of a Multipotent Self-Renewing Stromal Progenitor Population during Mammalian Kidney Organogenesis

SummaryThe mammalian kidney is a complex organ consisting of multiple cell types. We previously showed that the Six2-expressing cap mesenchyme is a multipotent self-renewing progenitor population for the main body of the nephron, the basic functional unit of the kidney. However, the cellular mechanisms establishing stromal tissues are less clear. We demonstrate that the Foxd1-expressing cortical stroma represents a distinct multipotent self-renewing progenitor population that gives rise to stromal tissues of the interstitium, mesangium, and pericytes throughout kidney organogenesis. Fate map analysis of Foxd1-expressing cells demonstrates that a small subset of these cells contributes to Six2-expressing cells at the early stage of kidney outgrowth. Thereafter, there appears to be a strict nephron and stromal lineage boundary derived from Six2-expressing and Foxd1-expressing cell types, respectively. Taken together, our observations suggest that distinct multipotent self-renewing progenitor populations coordinate cellular differentiation of the nephron epithelium and renal stroma during mammalian kidney organogenesis.

Intrinsic CXCR5 expression is required to maintain long-lived antigen specific CD4 T cells during chronic Mycobacterium tuberculosis infection (P3013)

Abstract Chronic antigenic stimulation is known to cause T cell functional exhaustion and death. During Mtb infections, antigen specific CD4 T cells are subject to chronic antigenic stimulation but a sizeable population of these cells survives and maintains the ability to produce IFN-γ. How these cells are maintained is not yet understood. Mtb-specific CD4 T cells can be classified based on the surface expression of the inhibitory receptors PD1 and KLRG1. The PD1+ cells proliferate robustly but produce low amounts of IFN-γ while KLRG1+ cells proliferate less but produce large amounts of IFN-γ. Significantly, the PD1+ cells are capable of differentiating into KLRG1+ cells and not vice versa indicating that a self-renewing progenitor population may be contained within the PD1+ cells. On further characterization we have found that PD1+ cells are heterogeneous containing three different subsets based on expression of the chemokine receptor CXCR5 i.e. PD1+CXCR5hi cells that resemble Tfh cells, PD1+CXCR5int and PD1+CXCR5lo cells. We found that B cells were not required to maintain the Tfh like cells or overall Mtb-specific CD4 T cells. However, using mixed bone marrow chimeras we found that intrinsic CXCR5 expression on CD4 T cells is required to maintain Mtb-specific CD4 T cells. Taken together, these studies indicate that during Mtb infections, a subset of CD4 T cells capable of self-renewal is induced and intrinsic CXCR5 is important for its maintenance.

Dynamics of dendritic cell development from precursors maintained in stroma-dependent long-term cultures.

Two distinct subsets of dendritic cells are produced within the non-adherent cell population of the stroma-dependent long-term culture system. These are the small subset containing dendritic cell precursors and their progeny, large long-term culture-dendritic cells, which resemble immature CD11c+CD11b+MHCIIloCD8alpha- dendritic cells. The replicative and developmental potential of cells produced in long-term culture were investigated as a model for production of dendritic cells from progenitors. Cell proliferation and apoptosis were examined by labelling with bromodeoxyuridine and Annexin-V, respectively. The developmental potential of cells was analysed following transfer on to stromal monolayers or into in vitro colony and transwell assays. Results demonstrate that small long-term culture-dendritic cells are stromal cell-dependent. In the absence of stroma, they become apoptotic and die. Furthermore, direct contact with stromal cells is necessary for the differentiation and proliferation of small precursor cells. The small cell subset contains no long-term self-renewing cells, but instead appears to contain cells committed to developing into large long-term culture dendritic cells. The large long-term culture dendritic cell subset also contains dividing cells. Survival of large long-term culture-dendritic cells is dependent on soluble stroma-derived factor(s) and not direct contact with the stromal layer. All data suggest that the long-term culture system supports dendritic cell development from a self-renewing progenitor population resident within the stroma that gives rise to committed dendritic cell precursors and immature dendritic cells.

Conditional Clara cell ablation reveals a self-renewing progenitor function of pulmonary neuroendocrine cells

The neuroepithelial body (NEB) is a highly dynamic structure that responds to chronic airway injury through hyperplasia of associated pulmonary neuroendocrine (PNE) cells. Although NEB dysplasia is correlated with preneoplastic conditions and PNE cells are thought to serve as a precursor for development of small cell lung carcinoma, mechanisms regulating expansion of the PNE cell population are not well understood. Based on studies performed in animal models, it has been suggested that NEB-associated progenitor cells that are phenotypically distinct from PNE cells contribute to PNE cell hyperplasia. We have previously used a Clara cell-specific toxicant, naphthalene, to induce airway injury in mice and have demonstrated that naphthalene-resistant Clara cells, characterized by their expression of Clara cell secretory protein (CCSP), and PNE cells contribute to airway repair and associated hyperplasia of NEBs. This study was conducted to define the contribution of NEB-associated CCSP-expressing progenitor cells to PNE cell hyperplasia after Clara cell ablation. Transgenic (CCtk) mice were generated in which herpes simplex virus thymidine kinase was expressed within all CCSP-expressing cells of the conducting airway epithelium through the use of transcriptional regulatory elements from the mouse CCSP promoter. Chronic administration of ganciclovir (GCV) to CCtk transgenic mice resulted in selective ablation of CCSP-expressing cells within conducting airways. Proliferation and hyperplasia of PNE cells occurred in the absence of detectable proliferation among any other residual airway epithelial cell populations. These results demonstrate that PNE cells function as a self-renewing progenitor population and that NEB-associated Clara cells are not necessary for PNE cell hyperplasia.