PDF电子书：Atlas of Human Pluripotent Stem Cells Derivation and Culturing

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* K- L( Z% A* r- p( LSpringer出版社的新书，很多图，对做人多能干细胞的童鞋可能有用。
* ~, G5 H& p& E" I5 _ISBN 978-1-61779-547-3 e-ISBN 978-1-61779-548-0$ h. A+ B, [. u
DOI 10.1007/978-1-61779-548-0' p4 p! H/ z7 h
Springer New York Dordrecht Heidelberg London
; s* d( Y5 N" aLibrary of Congress Control Number: 2011941608
2 [2 z2 C9 o$ T# [( N+ a/ x© Springer Science+Business Media, LLC 2012
6 B( ~9 [) Z# A" H[hide][/hide]2 d# ]9 F* w6 W( }! W
Contents：6 b  s8 v# M- J) C# V/ [, U: l7 U
1Methods for the Derivation of Human Embryonic- }0 n& A' q  [& u
Stem Cell Lines .......................................................................................... 1
2 I1 ^4 l+ b5 U6 q. ~1.1 Introduction ........................................................................................ 1
  B' H2 h" {0 ]! A4 i: e" c8 P1.2 Materials for ESC Line Derivation .................................................... 9* x8 D5 z4 f- T, v$ @7 j
1.3 Methods for hESC Isolation .............................................................. 97 k( R9 ^% ^! q4 N( u: M
1.3.1 hESC Isolation by Immunosurgery........................................ 121 o0 K+ s, ^$ y! k2 G& ?
1.3.2 Mechanical Removal of Trophectoderm ................................ 12, P5 F3 R% J+ @0 M$ q
1.3.3 Whole Embryo Approach for ESC Line Derivation .............. 13" V2 Q% Y) N% S8 u+ B6 G. m$ A
References ................................................................................................... 13
: B# u$ h+ \: o  Y1 p" \2 Morphology of Human Embryonic and Induced Pluripotent) S6 i, ]# U, @# _4 t: R/ s# p2 r9 }
Stem Cell Colonies Cultured with Feeders ............................................. 15# B( Y* R* X) O! `% Y
2.1 Introduction ........................................................................................ 152 ~, ~& v) [+ K; U" h; K+ F
2.2 Materials ............................................................................................ 16( `# h* d  l* P; |$ v. }4 U7 A
2.2.1 For Mouse Embryonic Fibroblasts (MEFs)$ h. I1 D% Z8 F; t9 V" r* L' g. J
and Foreskin Fibroblasts (HFFs) ........................................... 16
5 r: m! w& G1 C1 r( K4 ~2.2.2 For hPSC Maintenance .......................................................... 17( I! g5 b4 l, L. f2 ^9 v$ {
2.3 Methods ............................................................................................. 18
. b+ O6 P) {, s% r. o2.3.1 Feeder Culture Methods ........................................................ 18
: |3 J: u4 r& S4 V0 o: ]; D2.3.2 hPSC Culture ......................................................................... 226 M; B! F' I6 o; X5 M: c
References ................................................................................................... 386 Z- ^% p) U, ~: x3 ?2 }: H
3 Morphology of Human Embryonic Stem Cells and Induced" Q( H& I) J; [9 Q* y
Pluripotent Stem Cells Cultured in Feeder
/ ~$ J& J7 ^+ `/ r8 o9 n# aLayer-Free Conditions .............................................................................. 41
2 c3 o& Q1 t9 D5 O. J/ W3.1 Introduction ........................................................................................ 41
* Z% ?  g* c, m% n3.2 Materials for Feeder Layer-Free Culture of hPSCs ........................... 43
4 K8 w1 M' H1 ?0 S, [$ ?# T3.2.1 Matrix Preparation ................................................................. 43
% R- g# `8 Y( R' y+ P3.2.2 Culture Medium ..................................................................... 44
1 @4 m5 P3 M. C9 o3.3 Methods for hPSC Feeder Layer-Free Culture .................................. 44' Z7 |% P4 T: h# \2 h" z4 m6 S
3.3.1 Preparation of Matrix-Covered Plates ................................... 449 X% x+ a7 \' S# P; a
3.3.2 Splitting, Freezing, and Thawing hPSCs ............................... 450 q) O/ |- e' s' c6 A! \
3.3.3 Adaptation of PSCs to Feeder-Free Culture .......................... 45
8 ?& B6 W& }" N6 L3.3.4 Routine Culture of hPSCs ...................................................... 46
$ e+ r3 `! ~/ d- RReferences ................................................................................................... 54
; I  i$ j% S5 `* j4 Morphology of Undifferentiated Human Embryonic
, I' d, x. h( x0 v2 J0 {: |and Induced Stem Cells Grown in Suspension" s: u! C# w* p0 K
and in Dynamic Cultures .......................................................................... 575 ^8 O' Z5 R% x5 \) T1 [) y
4.1 Introduction ........................................................................................ 57
& Y+ d0 Y2 ]" i% d- K0 f4.2 Materials for Suspension Culture of hPSCs ...................................... 58) C& \* Z9 c4 h
4.2.1 Culture Medium ..................................................................... 58
0 c5 w$ z! o6 m7 X4.2.2 Splitting Medium ................................................................... 59
( h/ N6 g* |! @  N$ r/ Z5 O4.2.3 Freezing Medium ................................................................... 59
$ [% e9 t9 O$ M! C: `9 k' \4.3 Methods for Suspension Culture of hPSCs ....................................... 60
0 Q( [) m) K% N; H& _0 f4.3.1 Creating a hPSC Suspension Culture .................................... 60/ i+ a% x+ `! T' C( e( _0 `
4.3.2 Splitting hPSCs in Suspension ............................................... 60
9 L4 b! e5 B4 {5 q0 y3 R! T4.3.3 Freezing hPSCs in Suspension .............................................. 621 h* v) m& C+ e# r8 r* D
4.3.4 Thawing hPSCs in Suspension .............................................. 64
; N0 D5 F+ A% K' X$ }& w4 ]3 m6 S& |4.3.5 Culturing hPSCs in a Dynamic System ................................. 64
/ W; C! S1 o, a9 q4.3.6 Routine Culture of hPSCs in Suspension .............................. 65
+ [8 @" b7 @! bReferences ................................................................................................... 71" Q4 {6 M( P6 g+ c& a" m
5 Differentiation of Pluripotent Stem Cells In Vitro:
  i6 m0 ]. |  P2 c% xEmbryoid Bodies ....................................................................................... 73
' F5 N+ {: [; z5.1 Introduction ........................................................................................ 73
- R& m( p% o7 o- I* T& A3 U5.2 Materials for EB Formation ............................................................... 757 y. |6 L( Y5 T# Q, g% ]
5.2.1 Culture Medium Supplemented with Serum ......................... 75' q0 S" I( P( ]' k% `2 S
5.2.2 Splitting Medium Based on Collagenase ............................... 76
+ `' l6 h$ |7 V& B2 q, A9 m5.3 Methods for EB Formation and Culture ............................................ 76, m, D" ^* [9 p9 ]
5.3.1 EB Formation ......................................................................... 76( q5 @, Q6 |7 ~) l  b# z% _
5.3.2 Routine Culture of EBs .......................................................... 76- p. J  b. o  L& e- F7 o
5.3.3 Culturing EBs in Spinner Flasks ............................................ 785 R+ y# g5 k) z
References ................................................................................................... 88
; E- w7 f- b) \8 k/ B6 Differentiation of Pluripotent Stem Cells In Vivo:
6 r3 J4 J! H% P( b1 g5 g, I5 PTeratoma Formation ................................................................................. 91
1 K/ q( W/ R5 l2 l6.1 Introduction ........................................................................................ 91
; M+ V, E' ^8 F3 L* p6.2 Materials for Teratoma Formation ..................................................... 93  g) Q2 I, ?9 m8 b! Q% D# j
6.2.1 Culture Medium ..................................................................... 930 `+ e: I$ ?/ a$ i4 Y, R- t6 X
6.2.2 Syringe for Injecting Cells ..................................................... 93
1 s( p% H& |/ [( R5 F) S% D: Y6.3 Formation of Teratomas ..................................................................... 93
$ v. @! H6 k% v2 ]: ~: k5 W. p6.3.1 Protocol for Teratoma Formation .......................................... 93
' d$ @% b) T& I- I1 l+ b6.3.2 Routine Treatment of Mice and Teratoma ............................. 93
/ W2 a. Z5 y: m% m* g4 G, fReferences ................................................................................................... 103
9 t' \- c% J0 ~$ T9 N8 T" c' S7 Immunostaining ........................................................................................ 105
( t. S& B0 X) r. w" s7.1 Introduction ........................................................................................ 105' U4 ~' s  w  H9 l! v  O) H  p) e
7.2 Materials and Solutions for Immunostaining .................................... 111& k) a5 t9 K& K% Z: l" Y
7.2.1 Materials and Solutions for Immunohistochemistry
2 |+ Q4 I( r% r7 @" ^* Q+ Eof Paraffi n-Embedded Tissues ............................................... 1115 I- n$ k2 }4 |( Z6 l9 Y! D8 T
7.2.2 Materials and Solutions for Immunofl uorescence ................. 1119 P0 L4 O4 E1 u% h; Q
7.3 Immunostaining Procedures .............................................................. 1126 b) {, o9 ^( O7 b) }
7.3.1 Immunohistochemistry of Paraffi n-Embedded Tissues ......... 1127 G' y9 W9 ]: r& u
7.3.2 Immunofl uorescence of Cultured Cells ................................. 113
" |# E( V  w8 R; oReferences ................................................................................................... 1134 R. c& R6 q1 E( Y4 h, C
8 Karyotype and Fluorescent In Situ Hybridization- z) @! D% Z4 b- j. p. r/ R
Analysis of Human Embryonic Stem Cell and Induced) u8 B& j8 H. R& m* ^
Pluripotent Stem Cell Lines ..................................................................... 115! U/ r5 d+ B' _, [8 v% I8 G
8.1 Introduction ........................................................................................ 1154 M5 W- X3 _) D! P
8.1.1 Karyotype Analysis ............................................................... 115! T. u+ m4 d9 z
8.1.2 FISH Analysis ........................................................................ 121
! Y8 n. P5 J- }7 R: Y1 a8.2 Materials for Harvesting Cells for Karyotyping
8 Y2 Q/ h2 x6 ]) Land FISH Analysis ............................................................................. 124
$ L% l1 p+ z" u7 E8.2.1 Reagents ................................................................................. 1241 F! A4 Y$ ^2 D: }  D4 \6 F  ]
8.2.2 Solutions ................................................................................ 1246 m# @$ ^% K$ v
8.3 Procedure of Harvesting Cells for Karyotyping
4 Y! u: f0 T3 Q0 E2 h7 Vand FISH Analysis ............................................................................. 124
( [$ B% a( |- k3 J' y; yReferences ................................................................................................... 126
* q* s+ L' |) _+ d* |9 Method for the Derivation of Induced Pluripotent
4 A0 q2 n% \3 e% `1 B. F' QStem Cells from Human Hair Follicle Keratinocytes ............................ 127' _5 w1 @. g% O& T0 c# D
9.1 Introduction ........................................................................................ 127
( j9 A0 ?3 N/ X9 I3 {: g9.2 Materials ............................................................................................ 129
6 c8 M. U3 U$ t( c6 c3 j, P: y9.2.1 NIH-3T3/293T Cells .............................................................. 129
3 o  p* D$ j+ H9.2.2 Keratinocyte Derivation from Plucked Hair Follicles ........... 129. w( g1 p: w5 Q. R& V
9.3 Methods ............................................................................................. 130& v. _; z2 O0 w1 K/ r! j2 g
9.3.1 NIH-3T3 and 293T Culture Methods .................................... 130% _" p, J* X. N# L+ ]: s7 B
9.3.2 Keratinocyte Culture Methods ............................................... 132
- m1 ?( I7 T+ b4 C% n9.3.3 Preparation of the STEMCCA Virus for Infection ................ 1334 p/ {; J6 d5 a; a% E* i
9.3.4 Derivation of iPSCs from Hair Keratinocytes ....................... 1341 J- Z7 c# `$ o
References ................................................................................................... 137