Friday, July 31, 2015
Saturday, July 25, 2015
Region of Interest (ROI) of digital images and machine learning
Digital image and machine learning: ROIを最初に決めなきゃ。ROIなければ進む意味がない。
自分の場合だったら、移植した肝での1,000枚染めを鏡検することなんだろうけど、人力の方が安いだろうね。何を見たいのかをはっきりさせないと意味がない。
Guidance for Digital Pathology Whole Slide Imaging Devices from FDA
Technical Performance Assessment of Digital Pathology Whole Slide Imaging Devices
---Draft Guidance for Industry and Food and Drug Administration Staff---
Friday, July 24, 2015
Message for "OpenTein format" from Prof Park
OpenTein now accepts "SVS" of Aperio, "NDPI" of Hamamatsu, "SCN" of Leica and generic "TIFF" file formats.
See the license policy of OpenTein from http://opentein.hgc.jp/
Sunday, July 19, 2015
in vivo pluripotency of SEES cells
Numbers in parenthesis: OpenTein number.
B. Epidermis with keratinization (4621).
C. Glomerulus-like structure (quasi-glomerulus).
D. Proper gastric glands.
E. Hepatocytes
F. Ganglion.
G. Tracheobronchial tissue-like structure with ciliated epithelium and glands.
H. Cartilage and bone (4621).
I. Glandular structure (quasi-pancreas) (4621).
J. Immature neuroepithelium.
K and L. Hair follicles (4107) K: low-power view, L: high-power view.
M. Retina and retinal pigmented epithelium (4621).
Possible malignancy
akihiroumezawa - FYP100-109
Malignant cell proliferation (left upper) and epidermis with keratinization (right).
Wednesday, July 15, 2015
Future issue
1.Frozen
A.Likely OK (survive!!)
B.Subcapsular implantation in the kidney.
2.HE observation
A.Ymzk and me.
3.Form: Drug form depends on "Frozen" or not.
A. Ample-like form due to contamination.
4.Residual insulin content
Monday, July 13, 2015
Difference between the Saeko method (S) and HAES SOP (H)
1.Period of EB formation
A. S: 10 days in KSR in 96-well dishes in iPSellon without bFGF.
B.H: 3 days (4 to 7 days) in XF(-) in 10-cell dishes or 96-well dishes.
2.Period of adhesion.
A. S: 7 days in XF32 .
B.H: a month in XF32.
XF(-): XF32 without the growth factors.
Saeko with Kenta and Takaaki
Spontaneous differentiation
EB formation by the Saeko method.
Adhesion on collagen for seven days.
Tic was differentiated by the Saeko method with adhesion on collagen for seven days.
Test
A. Morphology
B. qRT-PCR
C. in vivo (not done)
D. Paraffin-embedded slides
E. Immunocytochemistry (fluorescent)
F. EM
Cell
A.MRCiPSC#51 (intermediate)---number of tubes: appropriately 5.
B.EdomiPSC#31 (most efficient hepatic differentiation)---number of tubes: 5.
C.MRCiPSC#16 (least efficient)---number of tubes: appropriately 5.
D.MRCiPSC#25(Tic) (intermediate)
Repeating above experiments (July 13, 2015)
Friday, July 3, 2015
Thursday, July 2, 2015
GCP-based clinical trial
Essential persons for clinical trial.
In hospital:Principal investigator does.
CRC helps PI.
-----------------------
From industry:
Monitor checks CRF and chart.
Auditor checks whole process.
-----------------------
Reliability guarantee from authorities
Saeko method.
1. 1 x 10^4 cells per well in a 96-well dish are cultivated for EB formation
2. EB formation in the EB medium for 10 days
EB medium
Knockout DMEM (380 ml)
KSR (100 ml)
Pc/SM (5 ml)
GlutaMax (5 ml)
Sodium Pyruvate (5 ml)
MEM (5 ml)
beta-ME (0.5 ml)
Takaaki
Hep2044iPS#4
Takaaki method
ALB and AFP expression ten times
1. BA-iPSCs
TY uses Yu's iPSCs, Hep2044 form hepatocytes of BA.
A. Four lines (#2 to #5) from Hep2044 (BA).
See Yu site.
Hepatic differentiation of BA-iPSCs.
A. Saeko method.
B. HAES SOP.
pHAES-WCB (Starting material) -> pHAES10 -> EB96 -> HAES a.Propagation with MEF in KSR. b.Period of EB formation i.4 days in XF(-) (= EB medium) in 96-well dishes. c.Period of adhesion. i.28 to 35 days in XF32. |
C. Low molecules published in Stem Cell Reports.
D.Takaaki method (Saeko-modified method)
a.Propagation with MEF in iPSellon.
b.Period of EB formation
i.10 days in XF32 in 96-well dishes.
c.Period of adhesion.
i. 7 days in XF32. Test
A. Morphology
B. qRT-PCR
C. in vivo
D. Paraffin-embedded slides
Exome analysis and karyotypic analysis of BA-iPSCs will be performed.
2. Hep2013 BA-iPSCs (A) and FH-iPSCs (B).
A.CXY-37 BA-iPSC: Nine lines from Hep2013 (BA) with Xiao.
Ike-59 BA-iPSC by Yu.
Test as iPSC undifferentiated state.
Exome analysis and karyotypic analysis of BA-iPSCs will be performed.
Wednesday, July 1, 2015
Hiroka
gRNAs 1-4, 2-4, 3-4, and 4-1were successful on 293 cells.
Neural stem cells from EdomiPS#S31 cells were transfected with the 1-4, 2-4, 3-4, and 4-1 (ATM disrupting gRNA) the Crisper-Cas9 system. Transfection efficiencies were quite low by the lipofection method. Electroporation method will be examined. The condition are from Prof Takagi (ask Marina).
Radiation experiments should be done after successful disruption of the ATM gene in EdomiPS#S31 cells.
Ataxia Telangiectasia SNP analysis
http://blogs.yahoo.co.jp/akihiroumezawa/34178477.htmlThe SNP genotyping by SNP array data was uploaded to the ncbi web site (GSE47498: Increased X-ray sensitivity and sustained chromosomal stability in Ataxia Telangiectasia-derived induced pluripotent stem (AT-iPS) cells, GSM1151202: AT1OS cells, GSM1151203: ATiPS-262 cells at passage 17, GSM1151204: ATiPS-263 cells at passage 27, GSM1151205: ATiPS-264 cells at passage 25, GSM1151206: ATiPS-024 cells at passage 25).
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