Dendritic Cells Direct Specific Stimulation of Hematopoietic Stem/Progenitor Cells Generating a Potentially Therapeutic Cell Population
Authors: Porat Yael , Assa-Kunik Efrat , Belkin Michael , Bulvik Shlomo 
Published: October 2019

Abstract
Abstract 483

Background:
Recent data show that dendritic cells (DCs) are important component of stem cell niches in the bone marrow and spleen, and as such may have a role in stem/progenitor cell homeostasis and in pro- and anti-angiogenic processes (Gabrilovich, 1996; Dikov, 2005; Sozzani, 2007). For the first time we report a process in which human Hematopoietic Stem/Progenitor Cells (HSPC) are specifically stimulated by activated DCs. This newly developed process makes it possible to use even unmobilized blood cells as a source for sufficient numbers of potentially therapeutic stem/progenitor cells, thus eliminating the need for surgical bone marrow harvesting and G-CSF mobilization.

Goal:
To show that DCs can direct the generation of an Enriched Endothelial Progenitor Cell (EnEPC) population, which includes Endothelial Progenitor Cells (EPC) and HSPCs, addressed to treat blood vessel malfunction.

Methods:
Samples of 250 ml blood from both healthy and diabetic patients were collected under hospital's IRB (Bulvik 15/150109) and used as the cell source. Selected immature plasmacytoid and myeloid DCs were alternatively activated for 2–24 hours in order to induce pro-angiogenic signals before being co-cultured with HSPCs. Cultures of up to 66 hours resulted in the generation of EnEPC in a formulation named BC1. BC1 was tested in-vitro by FACS, tube formation, colony forming units (CFU) and cytokine secretion tests. In-vivo BC1 was tested in the hind limb ischemia model (Goto, 2006; Kang, 2009) of critical limb ischemia (CLI) in order to evaluate its therapeutic potential, dosing levels and bio-distribution following intramuscular transplantation (IM). The study applied a genetically modified SCID/Nude mice model supporting evaluation of both safety and efficacy of BC1 treatment. A 21-day controlled blinded experiment included a control medium group (N=10); unprocessed cells (PreBC1, N=5); two BC1 groups of 2.5×10^6/mouse, BC1-1 (N=10) cultured for 1day and BC1-3 (N=10) for 3 and a lower cell dose group of 0.5×10^6/Mouse BC1-31 (N=5).

Results:
DC directed BC1 containing 70 ±5×10^6 cells with a viability of 96.9±1.9% is composed of a mixture of 40.2±11.9% EPC (expressing Ulex-lectin and uptake of AcLDL, CD202b (Tie2), CD309 (VEGGFR-2; KDR), CD31 and VEGFR1) and 29.8±14.3% HSPC (co-expressing CD34 and the migration/homing marker CD184 /CXCR4-R). In-vitro functional tests demonstrated angiogenic and hematopoietic potential and secretion of IL-8, VEGF, and IL-10 but not TNF and IFN.
In-vivo BC1 was found efficient and safe in the hind-limb ischemia model. Evaluation of clinical signs revealed an improvement in limb function and score in all BC1 treated groups over the control medium group. BC1 treatment doubled the blood flow (BF) to the legs from an average of 23±5% after injury to an average of 51±3.1% on day 21 after treatment (p<0.005).

Conclusions:
The presented data show that activated DCs can direct in-vitro cellular interactions resulting in a potentially therapeutic EnEPC population after a short-term culture of HSPC. This process makes it possible to use unmobilized blood as the raw material for generating stem/progenitor cell products. The method described here is far safer for patients and much more convenient for clinicians compared to existing methods, such as G-CSF mobilization or bone marrow and fat cells harvesting.
Further research needs to be done in order to test the safety and efficacy of these cells in patients suffering from cardiovascular diseases and blood vessel malfunctions.

Disclosures:
Porat: BioGenCell: Employment, Equity Ownership, Research Funding; Laniado Hospital: Consultancy. Assa-Kunik:BioGenCell: Employment; Laniado Hospital: Employment. Belkin:BioGenCell: Consultancy, Equity Ownership.