胚胎干细胞&诱导多能干细胞.pdf

胚胎干细胞&诱导多能干细胞
Embryonic Stem Cells & induced Pluripotent Stem Cell(iPSC)
Bruce Yin
1 仅供内部使用
胚胎干细胞ES
一种高度未分化细胞,具有发育的全能性。
滋养层
内细胞团胚
胎
干
细
胚囊腔胞
早期胚胎(囊胚)
2 仅供内部使用
hES饲养层培养体系
小鼠胚胎成纤维
细胞作为饲养层
•动物源性成分可能引
起动物源性病原体或支
原体的污染,从而限制
了hES 细胞的临床应用
3 仅供内部使用
HS Signal
MycoProbe Amplification
CUL00B Alkaline
PhosphataseY
Anti-DIG-Fab
DIG D D
+
Biotin 16S rRNA
16S rRNA 16S rRNA
B B
Capture Detection
Hybridization Streptavidin
4 仅供内部使用
MycoProbe Assay: Sample Preparation
• Cell Lysates
– Dilute cell pellet in 1X Cell Lysis Diluent to obtain a final
concentration of 30,000 to 120,000 cells/ml
• Cell Culture Supernatants
– Dilute sample 1:10 in 1X Cell Lysis Diluent
5 仅供内部使用
MycoProbe Assay:
Interpretation of Results
OD value* Result
< Mycoplasma Negative
– Ambiguous**
> Mycoplasma Positive
*Background (Negative control) subtracted
**Culture 2 to 3 additional days and then retest. If sample is still
ambiguous, then it should be considered to be negative.
6 仅供内部使用
MycoProbe versus
Mycoplasma PCR Detection Kit
Mycoplasma Mycoprobe PCR Agar
Plating
A. laidlawii1 + + +
M. arginini + + +
M. fermentans + + +
M. hominis + + +
M. hyorhinis + + +
M. orale + + +
M. pirum + - +
M. salivarum + + +
1A. = Acholeplasma
7 仅供内部使用
hES饲养层培养体系
Gamma射线处理过的小鼠胚胎成纤维细胞
iMEF,Cat# PSC001
(3 x 107 cells total)
用途:维持hESCs未分化状态和增殖能力。
特点:经射线处理,并经支原体检测,避免了支原
体的污染
8 仅供内部使用
hES采用无饲养层培养体系分离和培养
(1)条件培养基(滋养层细胞制备而来)
MEF-Conditioned Media (Catalog # AR005)
or
Human Feeder Cell-Conditioned Media
(Catalog # AR007)
用途:维持hESCs未分化状态和增殖能力。
特点:经支原体检测,避免了支原体的污染
binant human FGF basic (R&D
Systems, Catalog # 233-FB) or tissue
culture grade FGF basic (R&D Systems,
Catalog # 4114-TC)
Cultrex® Reduced Growth Factor
Basement Membrane Extract (BME)
(Catalog # 3433-005-01)
9 仅供内部使用
hES无饲养层培养体系分离和培养
(2)限定培养基
无血清的培养液+蛋白质和生长因子
HESCO培养液=bFGF+ insulin + transferrin+ Wnt3a