Recombinant TAT-BMI-1 fusion protein induces ex vivo expansion of human umbilical cord blood-derived hematopoietic stem cells

Abstract

Transplantation of hematopoietic stem cells (HSCs) is a well-established therapeutic approach for numerous disorders. HSCs are typically derived from bone marrow or peripheral blood after cytokine-induced mobilization. Umbilical cord blood (CB) represents an appealing alternative HSC source, but the small amounts of the individual CB units have limited its applications. The availability of strategies for safe ex vivo expansion of CB-derived HSCs (CB-HSCs) may allow to extend the use of these cells in adult patients and to avoid the risk of insufficient engraftment or delayed hematopoietic recovery.Here we describe a system for the ex vivo expansion of CB-HSCs based on their transient exposure to a recombinant TAT-BMI-1 chimeric protein. BMI-1 belongs to the Polycomb family of epigenetic modifiers and is recognized as a central regulator of HSC self-renewal. Recombinant TAT-BMI-1 produced in bacteria was able to enter the target cells via the HIV TAT-derived protein transduction peptide covalently attached to BMI-1, and conserved its biological activity. Treatment of CB-CD34+ cells for 3 days with repeated addition of 10 nM purified TAT-BMI-1 significantly enhanced total cell expansion as well as that of primitive hematopoietic progenitors in culture. Importantly, TAT-BMI-1-treated CB-CD34+ cells displayed a consistently higher rate of multi-lineage long-term repopulating activity in primary and secondary xenotransplants in immunocompromised mice. Thus, recombinant TAT-BMI-1 may represent a novel, effective reagent for ex vivo expansion of CB-HSC for therapeutic purposes.

Antioxidant Small Molecule Compound Chrysin Promotes the Self-Renewal of Hematopoietic Stem Cells

“…The ex vivo expansion of UCB-derived HSCs is an increasingly popular technique to improve the therapeutic effect of HSCT. Among numerous strategies proposed, cytokines are the most fundamental amplification tool such as classic combination of SCF, TPO, Flt3-L, and interleukins (Pineault et al, 2011), others like Delta-1, Jaggad-1, SALL4, and TAT-BMI-1 (Codispoti et al, 2017). In addition, small molecules, which are basically found through mass screening, are effective agonists for in vitro amplification of UCB HSCs.…”

Repopulating hematopoietic stem cells from steady-state blood before and after ex vivo culture are enriched in the CD34+CD133+CXCR4low fraction

“…This suggests that ex vivo expansion can provide an adequate tool to produce enough hematopoietic stem and progenitor cells to constitute a single hematopoietic graft from the contents of only one or two steady-state leukapheresis collections. The efficiency of the expansion procedure could, most likely, be further improved by using new approaches, for example the TAT-protein transduction peptide fused to regulatory factors or inhibition of HOXB4 degradation, 59 – 62 which is the object of our ongoing work. Furthermore, the CD34 neg fraction containing immuno-competent cells (T and B lymphocytes) can be preserved and an appropriate dose of these cells injected either during transplantation or later, depending on the need for an allogeneic immuno-effect.…”

The stem cell-associated transcription co-factor, ZNF521, interacts with GLI1 and GLI2 and enhances the activity of the Sonic hedgehog pathway

“…DAOY cells treated with or without 200 nM SAG were processed for nuclear and cytoplasmic extracts 64 . Cells were scraped, re-suspended in hypotonic lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, protease inhibitors (P8849, Sigma) and phosphatase inhibitor cocktails 2 and 3 (P0044, P5726, Sigma) and incubated on ice for 20 min.…”