Primary culture of human MSCs
Bone marrow samples of myeloma patients and healthy donors were obtained after
informed consent. Bone marrow aspirates were obtained from sternum of healthy
donors, or from the iliac crest of myeloma patients. For hMSCs from healthy donors,
whole BM mononuclear cells were collected by density gradient centrifugation with
Ficoll-Hypaque (Nycomed) and cultured according to the method described
previously.1 For hMSCs from MM and MGUS patients, an additional CD138 MACS
separation was performed to remove the malignant plasma cells before the initial
culture. Prior to use in experiments, hMSCs were characterized by their fibroblast-like
morphology, immunophenotype (CD90+, CD73+, CD166+ and CD45-) and
differentiation ability towards adipocytes, osteoblasts and chondrocytes in specific
induction media. The study was approved by the local ethical committee. hMSCs at
passage 2 were used in this study.
Osteogenic differentiation induction
2×104 hMSCs were plated in 1.5mL growth medium in a 12-well plate. After
overnight incubation, osteogenic differentiation was induced with Osteogenesis
Induction Medium (Lonza) containing dexamethasone, ascorbate, glycerophosphate,
L-Glutamine, Pen/Strep and MCGS. Medium was replaced every three days. Alkaline
phosphatase (ALP) expression and calcium deposit were used as early and late
markers for osteogenesis and were detected by ALP and Alizarin Red Staining,
respectively.
Qualitative ALP staining
After culture in osteogenic induction medium for indicated time, cells were washed
with PBS, and BCIP/NBT (5-Bromo-4-chloro-3-indolyl phosphate/Nitroblue
tetrazolium, Sigma-Aldrich) liquid substrate was added to each well, followed by 60
min incubation in the dark. ALP positive cells can be visualized under the light
microscope (dark purple color).
Quantitative ALP activity measurement
For quantification of ALP activity, 2×103 hMSCs were cultured in osteogenic
induction medium in a 96-well plate. Three days later, cells were washed once with
PBS and 100µL Alkaline Phosphatase Yellow (pNPP) Liquid ELISA substrate
(Sigma-Aldrich) was added and incubated for 30 min in the dark. Finally, ALP
activity was analyzed by an ELISA reader at 415nm wavelength. Alkaline
phosphatase activity was normalized to total protein of each well to determine
alkaline phosphatase index (Alkaline phosphatase index = alkaline phosphatase
spectrophotometer reading/protein× 1000).
Alizarin Red S staining
After two weeks culture in osteogenic induction medium, cells were washed with PBS
and fixed with 10% paraformaldehyde (Merck) for 15 min at room temperature. After
washing, cells were stained with 40mM freshly Alizarin Red solution (PH=4.2) and
incubated for 10 min at room temperature with gentle shaking. Alizarin was aspirated
and the wells were washed at least 3 times before observation. Calcium deposits can
be visualized by their red color. To quantify the staining, cultures were destained
using 10% cetylpyridinium chloride (CPC) in 10mM sodiumphosphate, pH7.0, for
15min at room temperature. Aliquots of exacts were diluted 10-fold in 10% CPC
solution, and Alizarin Red S concentration was determined by absorbance
measurement at 562nM on a multiplate reader (Thermo-Labsystems, Leuven,
Belgium).
Notch1 knock-down by RNA interference
To knock-down Notch1 expression, hMSCs were transfected with FlexiTube
GeneSolution for Notch1 (GS4851, Qiagen, Germany). The FlexiTube GeneSolution
for Notch1 provides four non-overlapping Notch1 RNAi duplexes for this gene to
obtain high knock-down efficiency. These duplexes (final concentration: 20nM) were
transfected into the hMSC with Lipofectamine RNAiMAX (Invitrogen, USA)
according to the manufacturer’s protocol. AllStars Negative Control siRNA (Qiagen)
was used as negative control. The efficiency of Notch1 knock-down was evaluated by
real time PCR and FACS.
Real time PCR
Total RNA was isolated using Trizol (Invitrogen, USA) and RNeasy Mini Kit (Qiagen,
Germany), following the manufacturer's instructions. The concentration and purity of
RNA was determined by a NanoDrop ND-1000 spectrophotometer. cDNA was
synthesized using Thermo Scientific VersoTM cDNA synthesis kit (Thermo Scientific,
UK). Quantitative real-time PCR analysis was done using the iCycler (Bio-Rad
Laboratories, USA) using the SYBR GreenER qPCR SuperMix for iCycler
(Invitrogen, USA) according to manufacturer's instructions. The primer sequences
used are listed in supplementary table. Transcript levels were normalized to the
housekeeping gene β-actin and analyzed by the relative quantification 2-ΔΔCt method.
References
1. De Becker A, Van Hummelen P, Bakkus M, Vande Broek I, De Wever J, De
Waele M, et al. Migration of culture-expanded human mesenchymal stem cells
through bone marrow endothelium is regulated by matrix metalloproteinase-2 and
tissue inhibitor of metalloproteinase-3. Haematologica 2007;92:440-49.