The sinoatrial node regulates the heart rate throughout life. Failure of this primary pacemaker results in life-threatening, slow heart rhythm. Despite its critical function, the cellular and molecular composition of the human sinoatrial node is not resolved. Particularly, no cell surface marker to identify and isolate sinoatrial node pacemaker cells has been reported. Here we use single-nuclei/cell RNA sequencing of fetal and human pluripotent stem cell-derived sinoatrial node cells to reveal that they consist of three subtypes of pacemaker cells: Core Pacemaker, Sinus Venosus, and Transitional Cells. Our study identifies a host of sinoatrial node pacemaker markers including MYH11, BMP4, and the cell surface antigen CD34. We demonstrate that sorting for CD34+ cells from stem cell differentiation cultures enriches for sinoatrial node cells exhibiting a functional pacemaker phenotype. This sinoatrial node pacemaker cell surface marker is highly valuable for stem cell-based disease modeling, drug discovery, cell replacement therapies, and the targeted delivery of therapeutics to sinoatrial node cells in vivo using antibody-drug conjugates.
© 2024. The Author(s).

Alcohol-associated liver disease (ALD) is a prevalent liver disease, yet research is hampered by the lack of suitable and reliable human ALD models. Herein, we generated human adipose stromal/stem cell (hASC)-derived hepatocellular organoids (hAHOs) and hASC-derived liver organoids (hALOs) in a three-dimensional system using hASC-derived hepatocyte-like cells and endodermal progenitor cells, respectively. The hAHOs were composed of major hepatocytes and cholangiocytes. The hALOs contained hepatocytes and nonparenchymal cells and possessed a more mature liver function than hAHOs. Upon ethanol treatment, both steatosis and inflammation were present in hAHOs and hALOs. The incubation of hALOs with ethanol resulted in increases in the levels of oxidative stress, the endoplasmic reticulum protein thioredoxin domain-containing protein 5 (TXNDC5), the alcohol-metabolizing enzymes ADH1B and ALDH1B1, and extracellular matrix accumulation, similar to those of liver tissues from patients with ALD. These results present a useful approach for understanding the pathogenesis of ALD in humans, thus facilitating the discovery of effective treatments.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

Adipose-derived mesenchymal stem cells (ASCs) represent a valid therapeutic option for clinical application in several diseases, due to their ability to repair damaged tissues and to mitigate the inflammatory/immune response. A better understanding of the underlying mechanisms regulating ASC biology might represent the chance to modulate their in vitro characteristics and differentiation potential for regenerative medicine purposes. Herein, we investigated the effects of the demethylating agent 5-azacytidine (5-aza) on proliferation, clonogenicity, migration, adipogenic differentiation and senescence of ASCs, to identify the molecular pathways involved. Through functional assays, we observed a detrimental effect of 5-aza on ASC self-renewal capacity and migration, accompanied by actin cytoskeleton reorganization, with decreased stress fibers. Conversely, 5-aza treatment enhanced ASC adipogenic differentiation, as assessed by lipid accumulation and expression of lineage-specific markers. We analyzed the involvement of the Akt/mTOR, MAPK and Wnt/β-catenin pathways in these processes. Our results indicated impairment of Akt and ERK phosphorylation, potentially explaining the reduced cell proliferation and migration. We observed a 5-aza-mediated inhibition of the Wnt signaling pathway, this potentially explaining the pro-adipogenic effect of the drug. Finally, 5-aza treatment significantly induced ASC senescence, through upregulation of the p53/p21 axis. Our data may have important translational implications, by helping in clarifying the potential risks and advantages of using epigenetic treatment to improve ASC characteristics for cell-based clinical approaches.
Copyright © 2022 Ceccarelli, Gerini, Megiorni, Pontecorvi, Messina, Camero, Anastasiadou, Romano, Onesti, Napoli and Marchese.

Glial cell line-derived neurotrophic factor (GDNF) has neuroprotective effects and may be a promising candidate for regenerative strategies focusing on neurodegenerative diseases. As GDNF cannot cross the blood-brain barrier to potentially regenerate damaged brain areas, continuous in situ delivery with host cells is desired. Here, a non-viral Sleeping Beauty transposon was used to achieve continuous in vitro overexpression of GDNF in immune-privileged human adipose tissue-derived mesenchymal stromal cells (GDNF-tASCs). In addition, in vivo survival, tolerance, and effectiveness of transfected cells were tested in a very mild 6-hydroxydopamine (6-OHDA)-induced dopamine depletion rat model by means of intrastriatal injection on a sample basis up to 6 months after treatment. GDNF-tASCs showed vast in vitro gene overexpression up to 13 weeks post-transfection. In vivo, GDNF was detectable 4 days following transplantation, but no longer after 1 month, although adipose tissue-derived mesenchymal stromal cells (ASCs) could be visualized histologically even after 6 months. Despite successful long-term in vitro GDNF overexpression and its in vivo detection shortly after cell transplantation, the 6-OHDA model was too mild to enable sufficient evaluation of in vivo disease improvement. Still, in vivo immunocompatibility could be further examined. ASCs initially induced a pronounced microglial accumulation at transplantation site, particularly prominent in GDNF-tASCs. However, 6-OHDA-induced pro-inflammatory immune response was attenuated by ASCs, although delayed in the GDNF-tASCs group. To further test the therapeutic potential of the generated GDNF-overexpressing cells in a disease-related context, a follow-up study using a more appropriate 6-OHDA model is needed.
© 2022 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons Ltd.

Urine-derived stem cells (USCs) have been widely researched as a novel cell source for stem cell therapy, but their immunomodulatory characteristics remain to be investigated. This study aimed to characterize the immunomodulatory properties of human USCs.
Human USCs were isolated from fresh voiding urine samples from healthy male donors and expanded. Their cell surface markers were characterized by flow cytometry analysis and the telomerase activities for several USCs clones were determined. The immunosuppressive potential of USCs was evaluated by the performing the mixed lymphocyte reaction (MLR) [co-culture with peripheral blood mononuclear cells (PBMNCs)] and natural killer cells (NK) cytotoxicity assay. USCs cytokines release profile was determined by using human cytokine proteome array.
USCs exhibited high cell surface expression of embryonic/mesenchymal stem cells (MSCs) markers CD29, CD44, CD54, CD73, CD90, CD146, and CD166, while lacked expression of hematopoietic stem cell markers CD11, CD14, CD19, CD31, CD34, CD45, B cell marker CD79, and co-stimulatory factors CD80 and CD86, thus, exhibiting the phenotype of MSCs. MLR indicated that USCs significantly inhibited the proliferation of PBMNCs, as compared to that of the human smooth muscle cells (SMCs). In cell cytotoxicity assays, NK cells displayed less cytotoxicity against USCs than against bone marrow mesenchymal stem cells (BMSCs) and SMCs. Furthermore, upon PBMNCs stimulation, USCs secreted higher levels of immunomodulatory cytokines, including IL-6, IL-8, MCP-1, RANTES, GROα, and GM-CSF, compared to those of BMSCs, especially when directly contact mix-culture with PBMNCs.
USCs secreted immunoregulatory cytokines and possessed immunomodulatory properties, comparable to those of BMSCs.
2021 Translational Andrology and Urology. All rights reserved.