Benedetta Mazzinghi

Phenotypic and functional features of human Th17 cells

T helper (Th) 17 cells represent a novel subset of CD4+ T cells that are protective against extracellular microbes, but are responsible for autoimmune disorders in mice. However, their properties in humans are only partially known. We demonstrate the presence of Th17 cells, some of which produce both interleukin (IL)-17 and interferon (IFN)-γ (Th17/Th1), in the gut of patients with Crohns disease. Both Th17 and Th17/Th1 clones showed selective expression of IL-23R, CCR6, and the transcription factor RORγt, and they exhibited similar functional features, such as the ability to help B cells, low cytotoxicity, and poor susceptibility to regulation by autologous regulatory T cells. Interestingly, these subsets also expressed the Th1-transcription factor T-bet, and stimulation of these cells in the presence of IL-12 down-regulated the expression of RORγt and the production of IL-17, but induced IFN-γ. These effects were partially inhibited in presence of IL-23. Similar receptor expression and functional capabilities were observed in freshly derived IL-17–producing peripheral blood and tonsillar CD4+ T cells. The demonstration of selective markers for human Th17 cells may help us to understand their pathogenic role. Moreover, the identification of a subset of cells sharing features of both Th1 and Th17, which can arise from the modulation of Th17 cells by IL-12, may raise new issues concerning developmental and/or functional relationships between Th17 and Th1.

Role for Interferon‐γ in the Immunomodulatory Activity of Human Bone Marrow Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) inhibit the proliferation of HLA‐unrelated T lymphocytes to allogeneic stimulation, but the mechanisms responsible for this activity are not fully understood. We show here that MSCs suppress the proliferation of both CD4+ and CD8+ T lymphocytes, as well as of natural killer (NK) cells, whereas they do not have an effect on the proliferation of B lymphocytes. The antiproliferative effect of MSCs was not associated with any effect on the expression of cell‐activation markers, induction of cell apoptosis, or mimicry/enhancement of T regulatory cell activity. The suppressive activity of MSCs was not contact‐dependent and required the presence of interferon (IFN)‐γ produced by activated T cells and NK cells. Accordingly, even activated B cells became susceptible to the suppressive activity of MSCs in the presence of exogenously added IFN‐γ. The suppressive effect of IFN‐γ was related to its ability to stimulate the production by MSCs of indoleamine 2,3‐dioxygenase activity, which in turn inhibited the proliferation of activated T or NK cells. These findings suggest that the beneficial effect on graft‐versus‐host disease induced by in vivo coinfusion with the graft of MSCs may be due to the activation of the immunomodulatory properties of MSCs by T cell– derived IFN‐γ.

Isolation and Characterization of Multipotent Progenitor Cells from the Bowman’s Capsule of Adult Human Kidneys

Regenerative medicine represents a critical clinical goal for patients with ESRD, but the identification of renal adult multipotent progenitor cells has remained elusive. It is demonstrated that in human adult kidneys, a subset of parietal epithelial cells (PEC) in the Bowmans capsule exhibit coexpression of the stem cell markers CD24 and CD133 and of the stem cell-specific transcription factors Oct-4 and BmI-1, in the absence of lineage-specific markers. This CD24+CD133+ PEC population, which could be purified from cultured capsulated glomeruli, revealed self-renewal potential and a high cloning efficiency. Under appropriate culture conditions, individual clones of CD24+CD133+ PEC could be induced to generate mature, functional, tubular cells with phenotypic features of proximal and/or distal tubules, osteogenic cells, adipocytes, and cells that exhibited phenotypic and functional features of neuronal cells. The injection of CD24+CD133+ PEC but not of CD24-CD133- renal cells into SCID mice that had acute renal failure resulted in the regeneration of tubular structures of different portions of the nephron. More important, treatment of acute renal failure with CD24+CD133+ PEC significantly ameliorated the morphologic and functional kidney damage. This study demonstrates the existence and provides the characterization of a population of resident multipotent progenitor cells in adult human glomeruli, potentially opening new avenues for the development of regenerative medicine in patients who have renal diseases.

An Alternatively Spliced Variant of CXCR3 Mediates the Inhibition of Endothelial Cell Growth Induced by IP-10, Mig, and I-TAC, and Acts as Functional Receptor for Platelet Factor 4

The chemokines CXCL9/Mig, CXCL10/IP-10, and CXCL11/I-TAC regulate lymphocyte chemotaxis, mediate vascular pericyte proliferation, and act as angiostatic agents, thus inhibiting tumor growth. These multiple activities are apparently mediated by a unique G protein–coupled receptor, termed CXCR3. The chemokine CXCL4/PF4 shares several activities with CXCL9, CXCL10, and CXCL11, including a powerful angiostatic effect, but its specific receptor is still unknown. Here, we describe a distinct, previously unrecognized receptor named CXCR3-B, derived from an alternative splicing of the CXCR3 gene that mediates the angiostatic activity of CXCR3 ligands and also acts as functional receptor for CXCL4. Human microvascular endothelial cell line-1 (HMEC-1), transfected with either the known CXCR3 (renamed CXCR3-A) or CXCR3-B, bound CXCL9, CXCL10, and CXCL11, whereas CXCL4 showed high affinity only for CXCR3-B. Overexpression of CXCR3-A induced an increase of survival, whereas overexpression of CXCR3-B dramatically reduced DNA synthesis and up-regulated apoptotic HMEC-1 death through activation of distinct signal transduction pathways. Remarkably, primary cultures of human microvascular endothelial cells, whose growth is inhibited by CXCL9, CXCL10, CXCL11, and CXCL4, expressed CXCR3-B, but not CXCR3-A. Finally, monoclonal antibodies raised to selectively recognize CXCR3-B reacted with endothelial cells from neoplastic tissues, providing evidence that CXCR3-B is also expressed in vivo and may account for the angiostatic effects of CXC chemokines.

Regeneration of Glomerular Podocytes by Human Renal Progenitors

Depletion of podocytes, common to glomerular diseases in general, plays a role in the pathogenesis of glomerulosclerosis. Whether podocyte injury in adulthood can be repaired has not been established. Here, we demonstrate that in the adult human kidney, CD133+CD24+ cells consist of a hierarchical population of progenitors that are arranged in a precise sequence within Bowmans capsule and exhibit heterogeneous potential for differentiation and regeneration. Cells localized to the urinary pole that expressed CD133 and CD24, but not podocyte markers (CD133+CD24+PDX- cells), could regenerate both tubular cells and podocytes. In contrast, cells localized between the urinary pole and vascular pole that expressed both progenitor and podocytes markers (CD133+CD24+PDX+) could regenerate only podocytes. Finally, cells localized to the vascular pole did not exhibit progenitor markers, but displayed phenotypic features of differentiated podocytes (CD133-CD24-PDX+ cells). Injection of CD133+CD24+PDX- cells, but not CD133+CD24+PDX+ or CD133-CD24- cells, into mice with adriamycin-induced nephropathy reduced proteinuria and improved chronic glomerular damage, suggesting that CD133+CD24+PDX- cells could potentially treat glomerular disorders characterized by podocyte injury, proteinuria, and progressive glomerulosclerosis.

Toll-Like Receptors 3 and 4 Are Expressed by Human Bone Marrow-Derived Mesenchymal Stem Cells and Can Inhibit Their T-Cell Modulatory Activity by Impairing Notch Signaling

Bone marrow (BM)‐derived mesenchymal stem cells (MSCs) are multipotent, nonhemopoietic progenitors that also possess regulatory activity on immune effector cells through different mechanisms. We demonstrate that human BM‐derived MSCs expressed high levels of Toll‐like receptors (TLRs) 3 and 4, which are both functional, as shown by the ability of their ligands to induce nuclear factor κB (NF‐κB) activity, as well as the production of interleukin (IL)‐6, IL‐8, and CXCL10. Of note, ligation of TLR3 and TLR4 on MSCs also inhibited the ability of these cells to suppress the proliferation of T cells, without influencing their immunophenotype or differentiation potential. The TLR triggering effects appeared to be related to the impairment of MSC signaling to Notch receptors in T cells. Indeed, MSCs expressed the Notch ligand Jagged‐1, and TLR3 or TLR4 ligation resulted in its strong downregulation. Moreover, anti‐Jagged‐1 neutralizing antibody and N[N‐(3,5‐difluorophenacetyl‐l‐alanyl)]‐S‐phenylglycine t‐butyl ester (DAPT), an inhibitor of Notch signaling, hampered the suppressive activity of MSCs on T‐cell proliferation. These data suggest that TLR3 and TLR4 expression on MSCs may provide an effective mechanism to block the immunosuppressive activity of MSCs and therefore to restore an efficient T‐cell response in the course of dangerous infections, such as those sustained by double‐stranded RNA viruses or Gram‐negative bacteria, respectively.

CD14+CD34low Cells With Stem Cell Phenotypic and Functional Features Are the Major Source of Circulating Endothelial Progenitors

Endothelial progenitor cells (EPCs) seem to be a promising tool for cell therapy of acute myocardial infarction, but their nature is still unclear. We show here that EPCs obtainable from peripheral blood (PB) derive from the adhesion-related selection in culture of a subset of CD14+ cells, which, when assessed by the highly-sensitive antibody-conjugated magnetofluorescent liposomes (ACMFL) technique, were found to express CD34. These CD14+CD34low cells represented a variable proportion at individual level of CD14+ cells, ranging from 0.6% to 8.5% of all peripheral-blood leukocytes, and constituted the dominant population among circulating KDR+ cells. By using the ACMFL technique, virtually all CD14+ cells present in the bone marrow were found to be CD14+CD34low double-positive cells. EPCs, as well as purified circulating CD14+CD34low cells, exhibited high expression of embryonic stem cell (SC) markers Nanog and Oct-4, which were downregulated in a STAT3-independent manner when they differentiated into endothelial cells (ECs). Moreover, circulating CD14+CD34low cells, but not CD14+CD34− cells, proliferated in response to SC growth factors, and exhibited clonogenicity and multipotency, as shown by their ability to differentiate not only into ECs, but also into osteoblasts, adipocytes, or neural cells. The results of this study may reconcile apparently contradictory data of the literature, showing the generation of PB-derived EPCs from either CD34+ or CD14+ cells. We suggest that the use of this previously unrecognized population of circulating CD14+CD34low cells, which exhibit both phenotypic and functional features of SCs, may be useful in improving cell-based therapies of vascular and tissue damage.

Essential but differential role for CXCR4 and CXCR7 in the therapeutic homingof human renal progenitor cells

Recently, we have identified a population of renal progenitor cells in human kidneys showing regenerative potential for injured renal tissue of SCID mice. We demonstrate here that among all known chemokine receptors, human renal progenitor cells exhibit high expression of both stromal-derived factor-1 (SDF-1) receptors, CXCR4 and CXCR7. In SCID mice with acute renal failure (ARF), SDF-1 was strongly up-regulated in resident cells surrounding necrotic areas. In the same mice, intravenously injected renal stem/progenitor cells engrafted into injured renal tissue decreased the severity of ARF and prevented renal fibrosis. These beneficial effects were abolished by blocking either CXCR4 or CXCR7, which dramatically reduced the number of engrafting renal progenitor cells. However, although SDF-1–induced migration of renal progenitor cells was only abolished by an anti-CXCR4 antibody, transendothelial migration required the activity of both CXCR4 and CXCR7, with CXCR7 being essential for renal progenitor cell adhesion to endothelial cells. Moreover, CXCR7 but not CXCR4 was responsible for the SDF-1–induced renal progenitor cell survival. Collectively, these findings suggest that CXCR4 and CXCR7 play an essential, but differential, role in the therapeutic homing of human renal progenitor cells in ARF, with important implications for the development of stem cell–based therapies.

Characterization of Renal Progenitors Committed Toward Tubular Lineage and Their Regenerative Potential in Renal Tubular Injury

Recent studies implicated the existence in adult human kidney of a population of renal progenitors with the potential to regenerate glomerular as well as tubular epithelial cells and characterized by coexpression of surface markers CD133 and CD24. Here, we demonstrate that CD133+CD24+ renal progenitors can be distinguished in distinct subpopulations from normal human kidneys based on the surface expression of vascular cell adhesion molecule 1, also known as CD106. CD133+CD24+CD106+ cells were localized at the urinary pole of Bowmans capsule, while a distinct population of scattered CD133+CD24+CD106− cells was localized in the proximal tubule as well as in the distal convoluted tubule. CD133+CD24+CD106+ cells exhibited a high proliferative rate and could differentiate toward the podocyte as well as the tubular lineage. By contrast, CD133+CD24+CD106− cells showed a lower proliferative capacity and displayed a committed phenotype toward the tubular lineage. Both CD133+CD24+CD106+ and CD133+CD24+CD106− cells showed higher resistance to injurious agents in comparison to all other differentiated cells of the kidney. Once injected in SCID mice affected by acute tubular injury, both of these populations displayed the capacity to engraft within the kidney, generate novel tubular cells, and improve renal function. These properties were not shared by other tubular cells of the adult kidney. Finally, CD133+CD24+CD106− cells proliferated upon tubular injury, becoming the predominating part of the regenerating epithelium in patients with acute or chronic tubular damage. These data suggest that CD133+CD24+CD106− cells represent tubular‐committed progenitors that display resistance to apoptotic stimuli and exert regenerative potential for injured tubular tissue. STEM CELLS2012;30:1714–1725

Regenerative Potential of Embryonic Renal Multipotent Progenitors in Acute Renal Failure

Bone marrow-and adult kidney-derived stem/progenitor cells hold promise in the development of therapies for renal failure. Here is reported the identification and characterization of renal multipotent progenitors in human embryonic kidneys that share CD24 and CD133 surface expression with adult renal progenitors and have the capacity for self-renewal and multilineage differentiation. It was found that these CD24+CD133+ cells constitute the early primordial nephron but progressively disappear during nephron development until they become selectively localized to the urinary pole of Bowmans capsule. When isolated and injected into SCID mice with acute renal failure from glycerol-induced rhabdomyolysis, these cells regenerated different portions of the nephron, reduced tissue necrosis and fibrosis, and significantly improved renal function. No tumorigenic potential was observed. It is concluded that CD24+CD133+ cells represent a subset of multipotent embryonic progenitors that persist in human kidneys from early stages of nephrogenesis. The ability of these cells to repair renal damage, together with their apparent lack of tumorigenicity, suggests their potential in the treatment of renal failure.