Xiao-nong Wang - Academia.edu (original) (raw)

Papers by Xiao-nong Wang

Tissue engineering of replacement tissues and organs manufactured with stem cells and biocompatib... more Tissue engineering of replacement tissues and organs manufactured with stem cells and biocompatible scaffolds has become a today’s reality. Bone marrow harbours the two types of adult stem cells—haematopoietic stem cells (HSCs) and multipotential stromal cells (MSCs). Up to date, clinical use of MSCs mostly relies on culture expansion; however, current MSC culture-expansion protocols, although clinically permittable, do not guarantee the preservation of native MSC characteristics and yield cellular products of variable quality and potency, which could be accountable for varied treatment outcomes. As a consequence, new technologies have now emerged that are based on uncultured MSCs, of various degrees of purity, in clinical settings. This chapter will provide an overview on the current state of the art in the field of uncultured MSCs as cell therapy. We will discuss highly pertinent safety issues such as purity and the role of contaminating immune and non-immune cells. Parallels will...

Bone Marrow Transplantation, 2015

Introduction: Umbilical cord blood (CB) is particularly enriched with very immature endothelial p... more Introduction: Umbilical cord blood (CB) is particularly enriched with very immature endothelial progenitors (i.e. endothelial colony forming cells, ECFC) able to promote neo-vascularization in hind limb ischemia immunodeficient mice (Schwarz TM et al. Arterioscler Thromb Vasc Biol 2012;32:e136). We previously demonstrated that CB ECFCs show scarce HLA antigen expression (Nuzzolo et al. Blood Transfus. 2014, Suppl 1:s367-74) and do no express AB0 blood group antigens. Indeed, similarly to mesenchymal cells, also the utilization of CB ECFCs could be not HLA-restricted. Here, we evaluated the revascularization potential of CB ECFCs in hind limb ischemia mouse model utilizing immunocompetent recipients. Materials (or patients) and methods: ECFCs were obtained according to Ingram et al. (Blood 2004;104:2752) and utilized after the third passage. Unilateral hind limb ischemia was induced in C57/BL6 mice as described (Biscetti F et al. Diabetes. 2010; 59:1496). Animals (n ¼ 20) received intramuscular injection of CB ECFCs (1x10 6 cells in 50 mL of PBS) into the right thigh and calf. The control group (n ¼ 10) received only PBS. The laser Doppler perfusion analysis was performed at weekly intervals for 28 days. At weeks1 and 4 after surgery, mice were sacrificed and tissues stained with hematoxylineosin to evaluate the muscle fiber integrity score (from 0, i.e. no injury, to 100, i.e. maximum injury) by counting 50 fiber/ animal and scoring individual fibers as 0 (no injury), 1 (visible vacuolization), or 2 (disintegration) (Chan RK et al. Surgery 2006;139:236). The presence of CB ECFC injected cells was evaluated by immunohistochemistry using an anti-human CD31moAb (Dako). Plasma samples of sacrificed mice were analyzed for various angiogenic growth factors using the Bioplex pro-mouse cytokine panel at Bioplex cytomer (Bio-Rad). Results: Overall, no limb loss was observed in treated and control animals. Nevertheless, at laser doppler analysis, the mean blood flow rate in ECFC treated mice was higher than in controls. The response was as early as day 7 and persisted until day 28 (0.4240.28 at day 7, 0.6340.42 at day 14 and 1.1040.82 at day 28, respectively). Regarding inflammatory cell infiltration, no difference were found in samples obtained from treated and control mice. At days 7, and to lesser extent at day 28, treated mice showed significantly higher number of injured fibers than controls (Figure 1a). After 7 days from injection, several human CD31 þ cells were detectable in treated animals, whereas lower amount of human CD31 þ cells was detectable at day 28. Finally, we found that the ECFC injected mice exhibited a a higher secretion of VEGF, bFGF, IL18, CCL9 and LIF in response to ischemia (Figure 1b). Conclusion: Overall, these data show that injected CB ECFCS were not rejected in C57/BL6 mice and contribute to revascularization and tissue repair both directly and indirectly, by stimulating the production of growth factors and chemokines in the recipients. Our findings support the utilization of CB ECFCs as allogeneic cell therapy product for untreatable critical limb ischemia. Supported by Fondi di Ateneo UCSC 2014. Disclosure of Interest: None declared. P382 A pilot study of post-transplant hypomethylating agents þ Bortezomib þ NK/gamma-delta T cell infusions in children with hematologic malignancies

MicroRNA levels quantified in whole blood varies from PBMCs [version 2; referees: 1 approved with... more MicroRNA levels quantified in whole blood varies from PBMCs [version 2; referees: 1 approved with reservations, 1 not approved]

A human in vitro skin explant test was developed by Alcyomics for the prediction of adverse immun... more A human in vitro skin explant test was developed by Alcyomics for the prediction of adverse immune reactions and assessment of relative potency. The skin explant test has a unique readout of histological damage, which ranges in severity from grade 0 and I (negative reaction) to grades II, III and IV damage (positive reaction) in human full-thickness skin. The test has been assessed for predicting responses to sensitizers and non-sensitizers, originally tested in the mouse local lymph node assay (LLNA). Results compared with the LLNA gave 95% specificity, 95% sensitivity and 95% concordance with a correlation coefficient of 0.9. Similar specificity and sensitivity were achieved for comparison of results to published human data with a correlation coefficient of 0.91. The test can also identify chemicals mis-classified in the mouse local lymph node assay (LLNA). Results of the skin test correlate with high or low T cell proliferation and IFNγ production, and these assays can be used as...

bioRxiv, 2020

The human skin confers biophysical and immunological protection through a complex cellular networ... more The human skin confers biophysical and immunological protection through a complex cellular network that is established early in development. We profiled ~500,000 single cells using RNA-sequencing from healthy adult and developing skin, and skin from patients with atopic dermatitis and psoriasis. Our findings reveal a predominance of innate lymphoid cells and macrophages in developing skin in contrast to T cells and migratory dendritic cells in adult skin. We demonstrate dual keratinocyte differentiation trajectories and activated cellular circuits comprising vascular endothelial cells mediating immune cell trafficking, disease-specific clonally expanded IL13/IL22 and IL17A/F-expressing lymphocytes, epidermal IL23-expressing dendritic cells and inflammatory keratinocytes in disease. Our findings provide key insights into the dynamic cellular landscape of human skin in health and disease. One Sentence Summary Single cell atlas of human skin reveals cell circuits which are quantitative...

Blood, 2014

Promising results from murine models and early stage clinical trials have shown that adoptive tra... more Promising results from murine models and early stage clinical trials have shown that adoptive transfer of regulatory T cells (Treg) prevents graft-versus-host disease (GvHD). However, the primary target of Treg mediated protection against GvHD is yet to be fully defined. We have previously shown that the presence of Treg during effector T cell priming is able to ameliorate cutaneous GvH reactions in vitro by blocking effector cell migration. This has led to the hypothesis that Treg modulation of dendritic cells (DC) could be a key mechanism by which Treg exert their protective role in GvHD. DC are fundamental for the initiation of allo-reactive immune responses and are critical in GvHD pathogenesis. We investigated the effect of Treg on the phenotypic profile and allo-reactive functions of DCs. Furthermore, the impact of Treg treatment on the ability of DCs to induce GvH target tissue damage was examined for the first time using an in vitro human GvHD skin explant model. Immature, m...

Blood, 2015

Dendritic cells (DC) play a key role in the pathogenesis of Graft versus Host Disease (GvHD), a c... more Dendritic cells (DC) play a key role in the pathogenesis of Graft versus Host Disease (GvHD), a complication of haematopoietic stem cell transplantation and offer an attractive target for therapy. Regulatory T cells (Treg) have a potent immunoregulatory effect on the maturation and the antigen-presenting cell (APC) function of DC and adoptive transfer of Treg is highly efficacious in the induction of tolerance in an experimental model of GvHD and has entered Phase I clinical trials. Several mechanisms of suppression have been proposed, including Treg acting directly on DCs, attenuating their antigen-presenting and co-stimulatory functions by arresting their maturation. However, the molecular basis underpinning these effects in DCs remains ill-defined. We investigated the effect of Treg treatment on DCs by conducting gene expression profiling and confirmed the functional consequences using downstream assays. Immature, mature and Treg-treated DCs were generated from immuno-magnetic is...

Myelopoiesis is invariably present, and contributes to pathology, in animal models of graft versu... more Myelopoiesis is invariably present, and contributes to pathology, in animal models of graft versus host disease (GVHD). In humans, a rich inflammatory infiltrate bearing macrophage markers has also been described in histological studies. In order to determine the origin, functional properties and role in pathogenesis of these cells, we isolated single cell suspensions from acute cutaneous GVHD and subjected them to genotype, transcriptome and in vitro functional analysis. A donor-derived population of CD11c+CD14+ cells was the dominant population of all leukocytes in GVHD. Surface phenotype and nanostring gene expression profiling indicated the closest steady-state counterpart of these cells to be monocyte-derived macrophages. In GVHD, however, there was upregulation of monocyte antigens SIRPα and S100A8/9, and transcripts associated with leukocyte trafficking, pattern recognition, antigen presentation, and co-stimulation. Isolated GVHD macrophages stimulated greater proliferation a...

BMC Biomedical Engineering, 2019

Background: Mesenchymal stromal cells (MSCs) are widely used in clinical trials for bone repair a... more Background: Mesenchymal stromal cells (MSCs) are widely used in clinical trials for bone repair and regeneration. Despite previous evidence showing a prominent osteogenic potential of 2D cultured CD271 enriched MSCs, the osteogenic potential of CD271 enriched cells cultured on 3D scaffold is unknown. Apatite-wollastonite glass ceramic (A-W) is an osteoconductive biomaterial shown to be compatible with MSCs. This is the first study comparing the attachment, growth kinetics, and osteogenic potential of two MSC populations, namely heterogeneous plastic adherence MSCs (PA-MSCs) and CD271-enriched MSCs (CD271-MSCs), when cultured on A-W 3D scaffold. Results: The paired MSC populations were assessed for their attachment, growth kinetics and ALP activity using confocal and scanning electron microscopy and the quantifications of DNA contents and p-nitrophenyl (pNP) production respectively. While the PA-MSCs and CD271-MSCs had similar expansion and tri-lineage differentiation capacity during standard 2D culture, they showed different proliferation kinetics when seeded on the A-W scaffolds. PA-MSCs displayed a well-spread attachment with more elongated morphology compared to CD271-MSCs, signifying a different level of interaction between the cell populations and the scaffold surface. Following scaffold seeding PA-MSCs fully integrated into the scaffold surface and showed a stronger propensity for osteogenic differentiation as indicated by higher ALP activity than CD271-MSCs. Furthermore, A-W scaffold seeded uncultured non-enriched bone marrow mononuclear cells also demonstrated a higher proliferation rate and greater ALP activity compared to their CD271-enriched counterpart. Conclusions: Our findings suggest that CD271-positive enrichment of a population is not beneficial for osteogenesis when the cells are seeded on A-W scaffold. Furthermore, unselected heterogeneous MSCs or BM-MNCs are more promising for A-W scaffold based bone regeneration. This leads to a conclusion of broader clinical relevance for tissue engineering: on the basis of our observations here the osteogenic potential observed in 2D cell culture should not be considered indicative of likely performance in a 3D scaffold based system, even when one of the cell populations is effectively a subset of the other.

International Journal of Stem cell Research & Therapy, 2015

Transplantation, Jan 5, 2016

Graft-versus-host (GvH) disease (GvHD) remains a serious concern for patients undergoing antivira... more Graft-versus-host (GvH) disease (GvHD) remains a serious concern for patients undergoing antiviral cellular therapy. Despite the major improvements in cellular immunotherapy, the immunogenicity of virus-specific T cells has not yet been fully defined. This present study aims to examine how cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTLs) respond to allogeneic antigen stimulation and whether they give rise to GvHD target tissue damage. Cytomegalovirus-specific CTLs were isolated by the IFN-γ secretion assay (gamma-catch) from healthy seropositive volunteers and expanded in vitro. The levels of intracellular IFN-γ, cytotoxic activity, IFN-γ and granzyme B secretion, and CD25 expression were measured using flow cytometry (fluorescence-activated cell sorting). The ability of CMV-CTLs to induce GvHD target tissue damage was evaluated using the human in vitro skin explant assay (skin explant assay). Cytomegalovirus-specific CTLs responded specifically to CMV-phosphoprotein 65...

Frontiers in Immunology, 2016

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for numero... more Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for numerous hematological malignancies. However, acute graft-versus-host disease (aGVHD) is still the major complication causing mortality. MicroRNAs (miRNAs) play a significant role in inflammation and have potential as prognostic and diagnostic biomarkers. This study investigated the role of two immune-specific miRNAs (miR-146a and miR-155) as biomarkers for aGVHD incidence in the peripheral blood of allo-HSCT patients prior to disease onset. The study showed that miR-146a and its statistical interaction with miR-155 at day +28 were predictive of aGVHD incidence. Interestingly, the expression levels of miR-146a and miR-155 negatively correlated with the transcription factor, SPI1 (PU.1gene) mRNA expression.

Bone marrow transplantation, Jan 14, 2015

Graft versus host disease (GVHD) is a major complication of haematopoietic SCT (HSCT). A number o... more Graft versus host disease (GVHD) is a major complication of haematopoietic SCT (HSCT). A number of inflammatory cytokines/chemokines are implicated in GVHD and have been identified in numerous single centre studies as potential biomarkers for acute and/or chronic GVHD. In this study, we analysed candidate inflammatory biomarkers (B-cell activating factor (BAFF), interleukin 33 (IL-33), CXCL10 and CXCL11) in a two-centre study. Biomarkers were evaluated pre-transplant and at serial time points post transplant in acute and chronic GVHD patient sera with time-matched control samples from patients without GVHD. Further validation was performed using the human skin explant assay, clinical GVHD biopsies and mRNA expression analysis. BAFF was significantly increased pre-transplant. BAFF, IL-33, CXCL10 and CXCL11 showed increased levels in acute GVHD patient sera and high protein expression in grades II-III of the in vitro skin explant graft versus host reaction (GVHR) group. BAFF, CXCL10 a...

PLOS ONE, 2015

Introduction Current clinical trials utilize mesenchymal stromal cells (MSCs) expanded in culture... more Introduction Current clinical trials utilize mesenchymal stromal cells (MSCs) expanded in culture, however these interventions carry considerable costs and concerns pertaining to culture-induced losses of potency. This study assessed the feasibility of new clinical-grade technology to obtain uncultured MSC isolates from three human intra-osseous tissue sources based on immunomagnetic selection for CD271-positive cells. Materials and Methods MSCs were isolated from bone marrow (BM) aspirates or surgical waste materials; enzymatically digested femoral heads (FHs) and reamer irrigator aspirator (RIA) waste fluids. Flow cytometry for the CD45 −/low CD73 + CD271 + phenotype was used to evaluate uncultured MSCs before and after selection, and to measure MSC enrichment in parallel to colony forming-unit fibroblast assay. Trilineage differentiation assays and quantitative polymerase chain-reaction for key transcripts involved in bone regeneration was used to assess the functional utility of isolated cells for bone repair. Results Uncultured CD45 −/low CD271 + MSCs uniformly expressed CD73, CD90 and CD105 but showed variable expression of MSCA-1 and SUSD2 (BM>RIA>FH). MSCs were enriched over 150-fold from BM aspirates and RIA fluids, whereas the highest MSC purities were obtained from FH digests. Enriched fractions expressed increased levels of BMP-2, COL1A2, VEGFC, SPARC and CXCL12 transcripts (BM>RIA>FH), with the highest up-regulation detected for CXCL12 in BM (>1300-fold). Following culture expansion, CD271-selected MSCS were tri-potential and phenotypically identical to plastic adherence-selected MSCs.

Immunity, 2015

In the original publication of this article, the name of an author was inadvertently misspelled. ... more In the original publication of this article, the name of an author was inadvertently misspelled. The corrected author name is Diego Miranda-Saavedra. The spelling is now correct in the online version of the study. The authors apologize for the inconvenience.

Transplant Immunology, 2015

Biomarker Graft-versus-host disease (GVHD) can be a fatal complication of allogeneic stem cell tr... more Biomarker Graft-versus-host disease (GVHD) can be a fatal complication of allogeneic stem cell transplantation (allo-HSCT). GVHD can be classified as acute (aGVHD: up to 100 days) or chronic (cGVHD: after 100 days) based on the timepoint of disease occurrence. At present there are a limited number of biomarkers available for use in the clinic. Thus, the aim of this research was to evaluate the biomarker potential of the extensively studied Q8 Heat Shock Protein 70 family members (HSPA1A/HSPA1B and HSPA1L) at the messenger RNA (mRNA) level in acute and cGVHD patient cohorts. In the skin biopsies, HSPA1L mRNA expression was lower in patients with severe aGVHD (grades II-III) when compared to those with none or low grade aGVHD (grades 0-I) and normal controls. In whole blood, HSPA1L mRNA expression level was significantly (p = 0.008) up-regulated at 28 days post-transplant in cGVHD patients with a significant area under the curve (AUC = 0.773). In addition, HSPA1B expression in whole blood was significantly higher at 3 months post-transplant in both the aGVHD grade II-III (p = 0.012) and cGVHD (p = 0.027) patients. Our initial results in this small cohort show that quantifying HSPA1L mRNA expression in the whole blood of allo-HSCT patients at day 28 post-allo-HSCT may be a useful predictive biomarker for cGVHD. 28

Transplantation Journal, 2012

Background. Regulatory T cells (Tregs) effectively ameliorate graft-versus-host disease (GVHD). T... more Background. Regulatory T cells (Tregs) effectively ameliorate graft-versus-host disease (GVHD). The mechanisms underlying Treg therapeutic effect on GVHD are not fully elucidated. This study investigates whether Treg prevention of GVH tissue damage is associated with blocking CD8 + effector T-cell tissue invasion, a question not yet addressed in humans. Method. Tissue-infiltrating T cells and histopathology scores were detected using an in vitro human GVHD skin explant model, together with immunohistochemistry, cytometric bead array, functional adhesion and migration assays, flow cytometry, and quantitative real-time polymerase chain reaction. Results. Treg intervention during priming significantly decreased effector T-cell infiltration into target tissue (PG0.01) resulting in a striking reduction in the histopathology score of tissue injury (PG0.0001). These results were coupled with reduced CXCR3 and cutaneous lymphocyte antigen expression by effector T cells, together with decreased CXCL10 and CXCL11 expression in target tissue. Treg intervention also impaired the functional interaction of CXCR3 and cutaneous lymphocyte antigen with their specific ligands (PG0.01) and suppressed the secretion of CXCL9, CXCL10, and interferon-F (PG0.01, PG0.05, and PG0.001, respectively). Late addition of Tregs into the effector phase abolished their ability to suppress effector T-cell tissue invasion, resulting in a total loss of their ability to ameliorate GVH tissue damage. Conclusion. Preventing effector T-cell tissue invasion is a critical mechanistic event leading to Treg attenuation of GVH tissue damage. This therapeutic effect is associated with a failure of CD8 + T cells to increase tissue homing receptors after allo-stimulation, together with a breakdown of interferon-FYinduced chemoattractant expression in the target tissue.

PLoS ONE, 2011

Background: The major histocompatibility complex (MHC) is the most important genomic region that ... more Background: The major histocompatibility complex (MHC) is the most important genomic region that contributes to the risk of graft versus host disease (GVHD) after haematopoietic stem cell transplantation. Matching of MHC class I and II genes is essential for the success of transplantation. However, the MHC contains additional genes that also contribute to the risk of developing acute GVHD. It is difficult to identify these genes by genetic association studies alone due to linkage disequilibrium in this region. Therefore, we aimed to identify MHC genes and other genes involved in the pathophysiology of GVHD by mRNA expression profiling. Methodology/Principal Findings: To reduce the complexity of the task, we used genetically well-defined rat inbred strains and a rat skin explant assay, an in-vitro-model of the graft versus host reaction (GVHR), to analyze the expression of MHC, natural killer complex (NKC), and other genes in cutaneous GVHR. We observed a statistically significant and strong up or down regulation of 11 MHC, 6 NKC, and 168 genes encoded in other genomic regions, i.e. 4.9%, 14.0%, and 2.6% of the tested genes respectively. The regulation of 7 selected MHC and 3 NKC genes was confirmed by quantitative real-time PCR and in independent skin explant assays. In addition, similar regulations of most of the selected genes were observed in GVHD-affected skin lesions of transplanted rats and in human skin explant assays. Conclusions/Significance: We identified rat and human MHC and NKC genes that are regulated during GVHR in skin explant assays and could therefore serve as biomarkers for GVHD. Several of the respective human genes, including HLA-DMB, C2, AIF1, SPR1, UBD, and OLR1, are polymorphic. These candidates may therefore contribute to the genetic risk of GVHD in patients.