Stem Cell Research Italy

XII meeting

PALERMO

June 13-15, 2024

The Long Non-coding RNA HOTAIR Controls the Selfrenewal, Cell Senescence, and Secretion of Anti-aging

Protein a-Klotho in Human Adult Renal Progenitor Cells

Angela Picerno1, Francesca Giannuzzi1, Claudia Curci2, Giuseppe De Palma3, Mariagiovanna Di Chiano4, Simona Simone2, Rossana Franzin2,5, Anna Gallone4,5, Vito Francesco Di Lorenzo6, Alessandra Stasi2,5, Giovanni Battista Pertosa2,5, Carlo Sabbà1, Loreto Gesualdo2,5, Fabio Sallustio1,5,*,

1Department of Interdisciplinary Medicine, University of Bari Aldo Moro, Bari, Italy

2Nephrology, Dialysis and Transplantation Unit, DETO, University of Bari Aldo Moro, Bari, Italy

3Institutional BioBank, Experimental Oncology and Biobank Management Unit, IRCCS Istituto Tumori “Giovanni Paolo II”, Bari, Italy, Bari, Italy

4Department of Basic Medical Sciences, Neuroscience and Sense Organs, University of Bari Aldo Moro, Bari, Italy

5MIRROR-Medical Institute for Regeneration, Repairing and Organ Replacement, Interdepartmental Center, University of Bari Aldo Moro, Bari, Italy

6Urology Unit, IRCCS Istituto Tumori “Giovanni Paolo II”, Bari, Italy

*Corresponding author: Fabio Sallustio, PhD, Department of Interdisciplinary Medicine, University of Bari Aldo Moro, Piazza G. Cesare,11 70124 Bari, Italy.

Email: fabio.sallustio@uniba.it

Abstract

The long non-coding RNAs (lncRNA) play an important role in several biological processes, including some renal diseases. Nevertheless, little is known about lncRNA that are expressed in the healthy kidneys and involved in renal cell homeostasis and development, and even less is known about lncRNA involved in the maintenance of human adult renal stem/progenitor cells (ARPCs) that have been shown to be very important for renal homeostasis and repair processes. Through a whole-genome transcriptome screening, we found that the HOTAIR lncRNA is highly expressed in renal progenitors and potentially involved in cell cycle and senescence biological processes. By CRISPR/Cas9 genome editing, we generated HOTAIR knockout ARPC lines and established a key role of this lncRNA in ARPC self-renewal properties by sustaining their proliferative capacity and limiting the apoptotic process. Intriguingly, the HOTAIR knockout led to the ARPC senescence and to a significant decrease in the CD133 stem cell marker expression which is an inverse marker of ARPC senescence and can regulate renal

tubular repair after the damage. Furthermore, we found that ARPCs expressed high levels of the a-Klotho anti-aging protein and especially 2.6-fold higher levels compared to that secreted by renal proximal tubular cells (RPTECs). Finally, we showed that HOTAIR exerts its function

through the epigenetic silencing of the cell cycle inhibitor p15 inducing the trimethylation of the histone H3K27. Altogether, these results shed new light on the mechanisms of regulation of these important renal cells and may support the future development of precision therapies for

kidney diseases.

Role of glycosphingolipid SSEA-3 and FGF2 in the stemness and lineage commitment of multilineage differentiating stress enduring (MUSE) cells

Domenico Aprile1, Nicola Alessio1, Tiziana Squillaro1, Giovanni Di Bernardo 1,2, Gianfranco Peluso3, Umberto Galderisi 1,2,4

1Department of Experimental Medicine,

Biotechnology and Molecular Biology Section,

University of Campania “Luigi Vanvitelli”,

Naples, Italy

2Sbarro Institute for Cancer Research and

Molecular Medicine, Center for Biotechnology,

Temple University, Philadelphia,

Pennsylvania, USA

3Faculty of Medicine and Surgery, Saint

Camillus International University of Health

Sciences, Rome, Italy

4Genome and Stem Cell Center (GENKOK),

Erciyes University, Kayseri, Turkey

Correspondence:

Umberto Galderisi, Department of

Experimental Medicine, Biotechnology and

Molecular Biology Section, University of

Campania “Luigi Vanvitelli”, Via Luigi De

Crecchio 7, Naples, Italy.

Email: umberto.galderisi@unicampania.it

Abstract

Objectives: Multilineage differentiating Stress Enduring (MUSE) cells are endogenous, stress-resistant stem cells, expressing pluripotency master genes and able to differentiate in cells of the three embryonic sheets. Stage-Specific Embryonic Antigen 3 (SSEA-3), a glycosphingolipid (GSL), is the marker for identifying MUSE cells and is used to isolate this population from mesenchymal stromal cells. GSLs modulate signal transduction by interacting with plasma membrane components. The growth factor FGF2, important for MUSE cells biology, may interact with GSLs. Specific cell surface markers represent an invaluable tool for stem cell isolation. Nonetheless their role, if any, in stem cell biology is poorly investigated. Functions of stem cells, however, depend on niche external cues, which reach cells through surface markers. We addressed the role of SSEA-3 in MUSE cell behaviour, trying to define whether SSEA-3 is just a marker or if it plays a functional role in this cell population by determining if it has any relationship with FGF2 activity.

Results: We evidenced how the SSEA-3 and FGF2 cooperation affected the self-renewal and clonogenic capacity of MUSE cells. The block of SSEA-3 significantly reduced the multilineage potential of MUSE cells with production of nullipotent clones.

Conclusions: We contributed to dissecting the mechanisms underlying MUSE cell properties for establishing successful stem-cell-based therapies and the promotion of MUSE cells as a tool for the in vitro disease model.

Co-Treatment of Erythroid Cells from b-Thalassemia Patients with CRISPR-Cas9-Based b39-Globin Gene Editing and Induction of Fetal Hemoglobin

Lucia Carmela Cosenza 1, Cristina Zuccato 1,2, Matteo Zurlo 1 , Roberto Gambari 2,* and Alessia Finotti 1,2,*

1 Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology,

University of Ferrara, 44121 Ferrara, Italy

2 Center ‘Chiara Gemmo and Elio Zago’ for the Research on Thalassemia, University of Ferrara,

44121 Ferrara, Italy

* Correspondence: gam@unife.it (R.G.); alessia.finotti@unife.it (A.F.); Tel.: +39-0532-974443 (R.G.);

+39-0532-974510 (A.F.)

In questo lavoro si dimostra che il Gene editing è una strategia efficace per correggere mutazioni genetiche in patologie quali la b-talassemia. Utilizzando il tool CRISPR-Cas9, gli autori hanno corretto la sequenza genica del gene mutato b39-globin, ottenendo una produzione de novo di emoglobina adulta (HbA).