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Impact of humanised isolation and culture conditions on stemness and osteogenic potential of bone marrow derived mesenchymal stromal cells.

ABSTRACT: Therapeutic potential of human bone marrow stromal/stem cells (hBMSC) must be developed using well defined xenogenic-free conditions. hBMSC were isolated from healthy donors (n?=?3) using different isolation and expansion methods. Donor I was isolated and expanded by either bone marrow directly seeded and cells expanded in 10% AB human serum (AB) +5?ng/ml fibroblast growth factor-2 (FGF2) [Direct(AB?+?FGF_low)] or Ammonium-Chloride-Potassium Lysing Buffer was used before the cells were expanded in 10% AB +5?ng/ml FGF-2 [ACK(AB?+?FGF_low)] or Lymphoprep density gradient medium was used before the cells were expanded in 10% AB +5?ng/ml FGF2 [Lympho(AB?+?FGF_low)] or bone marrow directly seeded and cells expanded in 10% pooled platelet lysate plasma (PL)?+?heparin (2 I/U/mL) [Direct(PL)]. Groups for donors II and III were: Direct(AB?+?FGF_low) or 10% AB +10?ng/ml FGF2 [Direct(AB?+?FGF_high)] or Direct(PL). HBMSCs were assessed for viability, multi-potency, osteogenic, inflammatory response and replicative senescence in vitro after 1 and 3 weeks. Pre-selected culture conditions, Direct(AB?+?FGF_high) or Direct(PL), were seeded on biphasic calcium phosphate granules and subcutaneously implanted in NOD/SCID mice. After 1 and 11 weeks, explants were analysed for inflammatory and osteogenic response at gene level and histologically. To identify implanted human cells, in situ hybridisation was performed. hBMSC from all conditions showed in vitro multi-lineage potency. hBMSCs expanded in PL expressed stemness markers in vitro at significantly higher levels. Generally, cells expanded in AB?+?FGF2 conditions expressed higher osteogenic markers after 1 week both in vitro and in vivo. After 11 weeks in vivo, Direct(AB?+?FGF_high) formed mature ectopic bone, compared to immature mineralised tissues formed by Direct(PL) implants. Mouse responses showed a significant upregulation of IL-1? and IL-1? expression in Direct(PL). After 1 week, human cells were observed in both groups and after 11 weeks in Direct(AB?+?FGF_high) only. To conclude, results showed a significant effect of the isolation methods and demonstrated a relatively consistent pattern of efficacy from all donors. A tendency of hBMSC expanded in PL to retain a more stem-like phenotype elucidates their delayed differentiation and different inflammatory expressions.

SUBMITTER: Suliman S

PROVIDER: S-EPMC6831606 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Impact of humanised isolation and culture conditions on stemness and osteogenic potential of bone marrow derived mesenchymal stromal cells.

Suliman Salwa S Ali Hassan R W HRW Karlsen Tommy A TA Amiaud Jerome J Mohamed-Ahmed Samih S Layrolle Pierre P Costea Daniela E DE Brinchmann Jan E JE Mustafa Kamal K

Scientific reports 20191105 1

Therapeutic potential of human bone marrow stromal/stem cells (hBMSC) must be developed using well defined xenogenic-free conditions. hBMSC were isolated from healthy donors (n = 3) using different isolation and expansion methods. Donor I was isolated and expanded by either bone marrow directly seeded and cells expanded in 10% AB human serum (AB) +5 ng/ml fibroblast growth factor-2 (FGF2) [Direct(AB + FGF<sub>low</sub>)] or Ammonium-Chloride-Potassium Lysing Buffer was used before the cells were ...[more]

PMID: 31690774

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Project description:Aging tissues present a progressive decline in homeostasis and regenerative capacities, which has been associated with degenerative changes in tissue-specific stem cells and stem cell niches. We hypothesized that amino acids could regulate the stem cell phenotype and differentiation ability of human bone marrow-derived mesenchymal stromal cells (hBMSCs). Thus, we performed a screening of 22 standard amino acids and found that D-tryptophan (10 μM) increased the number of cells positive for the early stem cell marker SSEA-4, and the gene expression levels of OCT-4, NANOG, and SOX-2 in hBMSCs. Comparison between D- and L-tryptophan isomers showed that the latter presents a stronger effect in inducing the mRNA levels of Oct-4 and Nanog, and in increasing the osteogenic differentiation of hBMSCs. On the other hand, L-tryptophan suppressed adipogenesis. The migration and colony-forming ability of hBMSCs were also enhanced by L-tryptophan treatment. In vivo experiments delivering L-tryptophan (50 mg/kg/day) by intraperitoneal injections for three weeks confirmed that L-tryptophan significantly increased the percentage of cells positive for SSEA-4, mRNA levels of Nanog and Oct-4, and the migration and colony-forming ability of mouse BMSCs. L-kynurenine, a major metabolite of L-tryptophan, also induced similar effects of L-tryptophan in enhancing stemness and osteogenic differentiation of BMSCs in vitro and in vivo, possibly indicating the involvement of the kynurenine pathway as the downstream signaling of L-tryptophan. Finally, since BMSCs migrate to the wound healing site to promote bone healing, surgical defects of 1 mm in diameter were created in mouse femur to evaluate bone formation after two weeks of L-tryptophan or L-kynurenine injection. Both L-tryptophan and L-kynurenine accelerated bone healing compared to the PBS-injected control group. In summary, L-tryptophan enhanced the stemness and osteoblastic differentiation of BMSCs and may be used as an essential factor to maintain the stem cell properties and accelerate bone healing and/or prevent bone loss.

Dataset Information

Impact of humanised isolation and culture conditions on stemness and osteogenic potential of bone marrow derived mesenchymal stromal cells.

Publications

Impact of humanised isolation and culture conditions on stemness and osteogenic potential of bone marrow derived mesenchymal stromal cells.

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