Pth 1 34

Human BM samples were obtained according to Institutional Review
Board (IRB)-approved protocol (MUG Graz IRB no. 19–252).
BM-mesenchymal stem cells (MSC) were isolated and expanded in
α-modified minimum essential medium (α-MEM; Sigma-Aldrich)
exchanging FBS with 10% pooled human platelet lysate (pHPL) (Reinisch et al., 2015 (link); Schallmoser et al., 2007 (link)). Purity of isolated MSC
was routinely tested by flow-cytometry following suggested guidelines (Dominici et al., 2006 (link)). Humanized
ossicles were formed as previously described (Reinisch et al., 2015 (link)). Briefly,
2×10⁶ BM-MSC were admixed with 300 μL of
matrigel-equivalent matrix (Angiogenesis assay kit, Millipore, MA) and
injected subcutaneously (4 injections per mouse) into the flanks of
6–12 week old female NSG mice. For 28 consecutive days (starting at
day +3) mice were treated with daily injections of human parathyroid
hormone (PTH [1–34]; R&D Systems, MN); 40
μg/kg body weight; dorsal neck fold) to further promote bone marrow
niche formation. 8–10 weeks post BM-MSC application, mice bearing
humanized ossicles were conditioned with 200 rads and hematopoietic cells
differentiated from AML-iPSCs with carrier C3H/10T1/2 cells were injected
directly intra-ossicle into 1–2 humanized ossicle-niches per mouse
(20μl). Assessment of AML-iPSC engraftment was performed
8–12 weeks post-transplantation.

Confluent BM-MSC were harvested using trypsin-based dissociation reagent (TrypLE Express, Thermo Fisher, Gibco). 2 × 10⁶ MSC were pelleted and resuspended in 60 μl of pure filter pHPL and admixed with 240 μL of matrigel-equivalent matrix (Angiogenesis assay kit, Millipore, Billerica, MA). Total volume of 300 μl matrix-cell mixtures was injected subcutaneously to generate humanized ossicle niches (up to four injections per mouse) into the flanks of 6 – 12 week old immunodeficient NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice (Jackson Laboratory, Bar Harbor, ME). Starting at day +3 – 7, mice received daily subcutaneous injections of human parathyroid hormone (PTH [1-34]; R&D Systems, Minneapolis, MN; 40 μg/kg body weight; dorsal neck fold) for 28 consecutive days to further promote bone marrow niche formation, as previously described.^{18 (link),19 (link)} Eight – 10 weeks post BM-MSC application the site of injection was shaved and transplants were evaluated for bone and marrow formation by palpation and by visual inspection (development of a purple hue is indicative of bone marrow niche formation).