Chondrogenic Differentiation and Analysis of MSC - PromoCell

Chondrogenic Differentiation
and Analysis of MSC
Application Note
Background
Mesenchymal Stem Cells (MSC) are
fibroblastoid multipotent adult stem cells
with a high capacity for self-renewal. So
far, these cells have been isolated from
several human tissues, e.g. bone marrow,
adipose tissue, umbilical cord matrix, ten-
don, lung, and the periosteum [1].
Recently it was shown that MSC recruit
from the perivascular niche which rep-
resents a tight network throughout the
vasculature of the whole body. These
perivascular cells lack endothelial/he-
matopoietic markers (CD31, CD34) but
express CD146, PDGF-R beta, and alka-
line Phosphatase [2].
Characterization
MSC show a CD10 + , CD13 + , CD44 + ,
CD73 + , CD105 + phenotype, but do not
express CD31 or CD45 [1]. In addition
to surface marker analysis, the most
common and reliable way to identify a
population of MSC is to verify their mul-
tipotency. MSC can be differentiated in
adipocytes, osteoblasts, myocytes, and
chondrocytes in vivo and in vitro [1,3].
In addition, trans-differentiation of MSC
into cells of non-mesenchymal origin, e.g.
hepatocytes, neurons and pancreatic islet
cells, has been observed in vitro when
specific culture conditions and stimuli
apply [1].
The directed differentiation of MSC is
carried out in vitro using the appropriate
differentiation media, e.g. the ready to
use PromoCell MSC Differentiation Me-
dia (see below for differentiation proto-
cols). These terminally differentiated cells
are histochemically stained to determine
their specific marker profile (see below
for staining protocol).

2
Application Note - Chondrogenic Differentiation and Analysis of MSC
Use aseptic techniques and a laminar flow bench.
Chondrogenic Differentiation
1. Seed Mesenchymal Stem Cells
Plate 1 x 10 5 MSC per well of a 96-well U-bottom suspension culture plate using
MSC Growth Medium. Work in duplicate.
2. MSC-spheroid formation
Spheroids will spontaneously form within 24 - 48 hours.
Note: The more cells you use, the bigger the spheroids. You may use up to 3 x 105 cells per well.
3. Induce MSC-spheroids
Induce one of the duplicate samples with MSC Chondrogenic Differentiation
Medium. Use MSC Growth Medium for the remaining well as a negative control.
4. Differentiate induced MSC-spheroids
Incubate for 21 days. Change Medium every third day. Be careful not to aspirate
the spheroids.
Chondrogenic
Differentiation

Important: Do not let the cells dry for longer than 30 sec.
throughout the entire staining procedure!
Detection of cartilage extracellular matrix
Chondrogenic differentiation of MSC in 3D spheroid culture results in formation of
cartilage with a typical extracellular matrix. A key molecule - besides collagen type II
within this extracellular matrix is the proteoglycan aggrecan. Aggrecan can be used as
an indicator for cartilage formation and can be detected with Alcian Blue, a dark-blue
copper-containing dye.
1. Prepare solutions and buffers
Mix 60 ml Ethanol (98 - 100%) with 40 ml Acetic Acid (98 - 100%). Dissolve
10 mg Alcian Blue 8 GX in this solution to achieve the Alcian Staining Solution. The
solution is stable for one year.
Mix 120 ml Ethanol (98 - 100%) with 80 ml Acetic Acid (98 - 100%) to get the
Destaining Solution.
2. Wash the cartilage spheroids
Take the cartilage spheroids from the incubator and carefully aspirate the medium.
Carefully wash the spheroids twice with Dulbecco’s PBS, w/o Ca ++ / Mg ++ (Cat. No.
C-40232).
Note: Do not aspirate the spheroids!
4. Fixation of the cartilage spheroids
Carefully aspirate the PBS and transfer the tissue culture dish to a fume hood.
Add enough neutral buffered formalin (10%) to cover the cartilage spheroids.
Incubate at room temperature for 60 min.
4. Wash the cartilage spheroids
Carefully aspirate the formalin and wash the spheroids twice with distilled water.
Note: Do not aspirate the spheroids!
5. Stain the cells
Carefully aspirate the distilled water and add enough Alcian Staining Solution to
generously cover the cartilage spheroids, as some evaporation will occur. Incubate
overnight at room temperature in the dark.
6. Wash the cells
Carefully aspirate the Alcian Staining Solution and wash the cartilage spheroids with
the Destaining Solution for 20 min. Repeat the wash step twice. Carefully aspirate
the Destaining Solution and add PBS.
Note: Cartilage will retain an intense dark-blue stain during the destaining procedure,
whereas other tissue will loose the blue color almost completely.
7. Analyze the cartilage spheroids
Cartilage is intense dark-blue, whereas other tissue is at most faintly bluish.
1)
2)
Please follow the recommended safety precautions for the chemicals used in this
procedure!
Application Note - Chondrogenic Differentiation and Analysis of MSC 3
Fig. 1: hMSC-BM spheroids after in vitro differentiation
into cartilage using PromoCell Mesenchymal Stem
Cell Chondrogenic Differentiation Medium (Alcian blue
staining). The induced spheroid exhibits a strong blue
staining for cartilage extracellular matrix (1). The control
spheroid cultured in Mesenchymal Stem Cell Growth
Medium is almost colorless (2).
Detection
of cartilage
extracellular
matrix

References
[1] da Silva Meirelles L, Caplan AI, Nardi NB., Stem Cells 2008; 26(9):2287-99.
[2] Crisan M, Yap S, Casteilla L, et al., Cell Stem Cell 2008; 3(3):301-13.
[3] Caplan AI. Cell Stem Cell 2008, 3(3):229-30.
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