Small molecule‐mediated modulation of ubiquitination and neddylation improves HSC function ex vivo

Abstract

Hematopoietic stem cells (HSCs) are particularly characterized by their quiescence and self-renewal. Cell cycle regulators tightly control quiescence and self-renewal capacity. Studies suggest that modulation of ubiquitination and neddylation could contribute to HSC function via cyclin-dependent kinase inhibitors (CDKIs). S-phase kinase-associated protein 2 (SKP2) is responsible for ubiquitin-mediated proteolysis of CDKIs. Here, we modulated overall neddylation and SKP2-associated ubiquitination in HSCs by using SKP2-C25, an SKP2 inhibitor, and MLN4924 (Pevonedistat) as an inhibitor of the NEDD8 system. Treatments of SKP2-C25 and MLN4924 increased both murine and human stem and progenitor cell (HSPC) compartments. This is associated with the improved quiescence of murine HSC by upregulation of p27 and p57 CDKIs. A colony-forming unit assay showed an enhanced in vitro selfrenewal potential post inhibition of ubiquitination and neddylation. In addition, MLN4924 triggered the mobilization of bone marrow HSPCs to peripheral blood.Intriguingly, MLN4924 treatment could decrease the proliferation of murine bone marrow mesenchymal stem cells or endothelial cells. These findings shed light on the contribution of SKP2, and associated ubiquitination and neddylation in HSC maintenance, self-renewal, and expansion.

BML‐260 promotes the growth of cord blood and mobilized peripheral blood hematopoietic stem and progenitor cells with improved reconstitution ability

“…Our criteria for the determination of the effective small molecules on HSPC expansion was based on the detection of greater than twofold increase in minimum two parameters, especially CD34 + cell and ALDH high populations (showed in Figure 2) which are commonly used for HSPC and HSCs, respectively. Taken together, we briefly determined that treatment with #2 (p300/CBP Inhibitor VI), #19 (2‐APB), #32 (BML‐260), #33 (TAME), #34 (MEISi‐1), and #35 (MEISi‐2) inhibitors among 35 HSMs highly induced enhancement of UCB‐HSPC content compared to DMSO and previously tested molecules 8 . Therefore, we selected untested compounds in addition to the previously tested control compounds for the next experiments to investigate their role in human HSC expansion.…”

“…Taken together, we briefly determined that treatment with #2 (p300/CBP Inhibitor VI), #19 (2-APB), #32 (BML-260), #33 (TAME), #34 (MEISi-1), and #35 (MEISi-2) inhibitors among 35 HSMs highly induced enhancement of UCB-HSPC content compared to DMSO and previously tested molecules. 8 Therefore, we selected untested compounds in addition to the previously tested control compounds for the next experiments to investigate their role in human HSC expansion.…”

“…7 Small molecules are chemicals with a low molecular weight (900 Da), are easily diffused across cell membranes, and are nonimmunogenic. 8 They are widely used due to their ability to control stem cell fate, proliferation, self-renewal, differentiation, and cell death. Because of their low cost, ease of manipulation, and control, they are also very valuable tools for improving therapeutic approaches.…”

“…However, these culture and expansion protocols did not completely ensure HSC self‐renewal 7 . Small molecules are chemicals with a low molecular weight (900 Da), are easily diffused across cell membranes, and are nonimmunogenic 8 . They are widely used due to their ability to control stem cell fate, proliferation, self‐renewal, differentiation, and cell death.…”

Biomedical polymer scaffolds mimicking bone marrow niches to advance in vitro expansion of hematopoietic stem cells

“…To date, various in vitro HSC culture methods have been developed to obtain more pluripotent HSCs. 8,9 Compared to traditional two-dimensional (2D) cell culture, three-dimensional (3D) cell culture represents a powerful method to the advancement and development of tissue engineering applications, as it plays an essential role in directing cell behaviors such as migration, proliferation and differentiation. [10][11][12] To reconstitute the features of tissue in the native 3D extracellular matrix (ECM), many types of scaffold biomaterials, including fiber, graphene, and hydrogel, have been extensively developed for their excellent biocompatibility, favourable biodegradability, controllable physical and chemical properties, and accessible presentation of biological cues.…”