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

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7 G4 h: H1 w- [$ B$ s4 fSpringer出版社的新书，很多图，对做人多能干细胞的童鞋可能有用。
% Y7 O1 F2 v& l1 w0 S( DISBN 978-1-61779-547-3 e-ISBN 978-1-61779-548-0
$ q" Z- v8 x3 s" X; ?) r# ?DOI 10.1007/978-1-61779-548-0+ {4 @/ j. P) B' J8 c' T% q( G$ a
Springer New York Dordrecht Heidelberg London
4 m- N, g. z4 W/ c; y  N- i" B2 ?Library of Congress Control Number: 2011941608
' m( n2 i6 B- O" _% v& ~) w- m! f© Springer Science+Business Media, LLC 2012* A2 K* Q" ~5 x0 z8 ^
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2 ?4 v- w7 Y, f7 U' N7 MContents：/ }* f. q0 d) E/ @/ s- M
1Methods for the Derivation of Human Embryonic# y: P6 [7 A4 H$ C$ ~6 M/ p4 z2 C
Stem Cell Lines .......................................................................................... 1& b- h2 V' |; W. t
1.1 Introduction ........................................................................................ 1/ g* j2 ~9 C# X- z' A
1.2 Materials for ESC Line Derivation .................................................... 9
4 v7 A, n( Z; i) }1.3 Methods for hESC Isolation .............................................................. 9
% A6 V3 c" @4 y( A: u! D1.3.1 hESC Isolation by Immunosurgery........................................ 123 D# t. P/ O! i
1.3.2 Mechanical Removal of Trophectoderm ................................ 12
) }3 K( {% t, K8 W- g1.3.3 Whole Embryo Approach for ESC Line Derivation .............. 13
- E+ x- g+ V1 eReferences ................................................................................................... 13
/ O0 v1 r) s( ~4 L2 Morphology of Human Embryonic and Induced Pluripotent  Y  B) L" _/ t4 i% |
Stem Cell Colonies Cultured with Feeders ............................................. 157 O5 A/ B& J+ I! a, a( A
2.1 Introduction ........................................................................................ 15  d0 a- ]) p4 n2 H6 D
2.2 Materials ............................................................................................ 16
9 I# h/ U& D/ d! p, Q. Z5 J) B2.2.1 For Mouse Embryonic Fibroblasts (MEFs)
3 m' T; }5 A8 T/ ?" D$ hand Foreskin Fibroblasts (HFFs) ........................................... 168 x( X* F0 z" \) j
2.2.2 For hPSC Maintenance .......................................................... 17
; I+ _( v" f: G0 g# d2.3 Methods ............................................................................................. 18/ W% C$ a5 E; F# ~
2.3.1 Feeder Culture Methods ........................................................ 18
5 A9 i& e+ w0 U4 e! O: j) q4 W7 A2.3.2 hPSC Culture ......................................................................... 22! b) L; _- J  _' ^4 i
References ................................................................................................... 38
6 M+ n9 O" o' G  S# n3 Morphology of Human Embryonic Stem Cells and Induced
9 ~* e- p* X! Q$ x( n+ IPluripotent Stem Cells Cultured in Feeder
1 y! I" }/ D* B8 ELayer-Free Conditions .............................................................................. 41; l7 ^, y  q9 W7 u0 a
3.1 Introduction ........................................................................................ 41
' `, |4 ~1 E+ n& h  V- ^! \3.2 Materials for Feeder Layer-Free Culture of hPSCs ........................... 43
" W7 x: e" O3 L; o  z. v$ y" V3.2.1 Matrix Preparation ................................................................. 43
0 I4 g! m* p7 x/ V& v, D3 p1 W3.2.2 Culture Medium ..................................................................... 44
  F7 e; [6 Q4 ?* E. T# C3.3 Methods for hPSC Feeder Layer-Free Culture .................................. 44' ^7 \6 F6 [1 C, _
3.3.1 Preparation of Matrix-Covered Plates ................................... 44
' r& G/ a4 \6 U: z* E7 f# u+ E3.3.2 Splitting, Freezing, and Thawing hPSCs ............................... 45
3 R( n4 o. s# z! h% E3.3.3 Adaptation of PSCs to Feeder-Free Culture .......................... 45
& s& j; l' t- g9 O. G7 E3.3.4 Routine Culture of hPSCs ...................................................... 463 Q- h! |7 M2 i0 c
References ................................................................................................... 54
. {4 [! T" D6 D4 s  V4 Morphology of Undifferentiated Human Embryonic
' ?5 \7 q3 T- J6 |3 _1 f! A  U9 ]and Induced Stem Cells Grown in Suspension/ o: d6 F$ q/ v7 T& M6 g
and in Dynamic Cultures .......................................................................... 57) P2 [! }) g% L, `
4.1 Introduction ........................................................................................ 57
8 w9 V5 u( n6 q# k9 n4.2 Materials for Suspension Culture of hPSCs ...................................... 58- n9 G0 h( Z& G& J, R8 M! o
4.2.1 Culture Medium ..................................................................... 582 l1 D& s3 a; j1 F6 H& Y
4.2.2 Splitting Medium ................................................................... 59+ L, d" F" W1 B8 h" c2 u8 i. F' D
4.2.3 Freezing Medium ................................................................... 59
/ i2 ^: R2 n9 S  {# n1 a3 }4.3 Methods for Suspension Culture of hPSCs ....................................... 60$ p4 T/ a' y1 F1 K1 u
4.3.1 Creating a hPSC Suspension Culture .................................... 60  `/ `7 ^1 N( ^3 q# C: J$ R0 Y7 V
4.3.2 Splitting hPSCs in Suspension ............................................... 602 }; I; ?, O3 R
4.3.3 Freezing hPSCs in Suspension .............................................. 62, _4 v% ?  o. M. I1 O, K- L
4.3.4 Thawing hPSCs in Suspension .............................................. 645 d$ u8 s; |- u8 Q$ h( N
4.3.5 Culturing hPSCs in a Dynamic System ................................. 64& j3 h7 |- B& u1 B* q
4.3.6 Routine Culture of hPSCs in Suspension .............................. 65' M; A; @0 ]8 z3 e
References ................................................................................................... 71
% p; @$ j- V1 {6 z5 Differentiation of Pluripotent Stem Cells In Vitro:
0 _6 O  p" u* l" e0 kEmbryoid Bodies ....................................................................................... 73
; w# I4 |+ _: |/ p5.1 Introduction ........................................................................................ 73: z8 X7 M/ d2 I1 Q
5.2 Materials for EB Formation ............................................................... 75
$ C, ]8 Y: x$ T' w1 V( K% n, f5.2.1 Culture Medium Supplemented with Serum ......................... 75
+ U$ d# M6 `5 Q8 w1 A) G5.2.2 Splitting Medium Based on Collagenase ............................... 766 n; a3 K1 X+ q. P8 ~
5.3 Methods for EB Formation and Culture ............................................ 76" Q4 N! Z0 d1 Y7 j! N7 s# i
5.3.1 EB Formation ......................................................................... 76
5 m; C. a& |* v. c+ Q8 [8 A1 z5.3.2 Routine Culture of EBs .......................................................... 760 ]! |8 H, n+ ^" D. q
5.3.3 Culturing EBs in Spinner Flasks ............................................ 78
- T0 [8 D1 I9 w/ f5 ]References ................................................................................................... 88
" X- u# @1 }0 v1 H  C, M! e! V6 Differentiation of Pluripotent Stem Cells In Vivo:
/ I+ u( o$ Q3 u* F- k% \# \: eTeratoma Formation ................................................................................. 91( K3 `6 G6 ~0 ~6 {3 G0 _
6.1 Introduction ........................................................................................ 91
/ u: ^; d  ?) ~6.2 Materials for Teratoma Formation ..................................................... 938 K: j  W, i7 x5 e0 e" t9 K
6.2.1 Culture Medium ..................................................................... 933 y6 y- ~( C0 N
6.2.2 Syringe for Injecting Cells ..................................................... 93
5 u; A2 H$ i, V/ `) l1 N6.3 Formation of Teratomas ..................................................................... 93/ p  C4 R% F( l$ Q: G8 U
6.3.1 Protocol for Teratoma Formation .......................................... 938 a/ R& T1 J, i  o
6.3.2 Routine Treatment of Mice and Teratoma ............................. 93
# h5 t! B- g3 T0 dReferences ................................................................................................... 1033 O3 v( f1 p* O
7 Immunostaining ........................................................................................ 1051 L' ~: X  w( U; ^+ i
7.1 Introduction ........................................................................................ 105
, S2 O+ u7 j0 c( N5 R7.2 Materials and Solutions for Immunostaining .................................... 111
; x  d4 R! |5 l7.2.1 Materials and Solutions for Immunohistochemistry
9 Q/ j( C( r7 ]of Paraffi n-Embedded Tissues ............................................... 111
$ s( W2 I0 n7 W+ O% k- s7.2.2 Materials and Solutions for Immunofl uorescence ................. 111
- {4 [4 E* b3 a# N8 Y9 {! g7.3 Immunostaining Procedures .............................................................. 1124 y( S' l! @2 L! W, m  Y3 {  L% o
7.3.1 Immunohistochemistry of Paraffi n-Embedded Tissues ......... 112  [7 `- D& W2 V' g
7.3.2 Immunofl uorescence of Cultured Cells ................................. 113
. |6 @# Y2 s1 vReferences ................................................................................................... 1131 f* j2 F$ D  `: G$ V& F6 f; w
8 Karyotype and Fluorescent In Situ Hybridization
( h- T0 Q2 f/ `Analysis of Human Embryonic Stem Cell and Induced
: B/ X6 T) u0 I7 r+ I2 Z( n. sPluripotent Stem Cell Lines ..................................................................... 1158 A- j" K" y! o$ R6 l
8.1 Introduction ........................................................................................ 115/ E1 L$ F6 g% w+ m, T- Z
8.1.1 Karyotype Analysis ............................................................... 115; s2 G  v7 o9 f0 L
8.1.2 FISH Analysis ........................................................................ 121/ a& o! [) B. }, Q4 W6 @
8.2 Materials for Harvesting Cells for Karyotyping3 H- I" M' k1 Q& n1 u5 M: g
and FISH Analysis ............................................................................. 124
+ g# d9 z( J" A/ K  {' s/ `8.2.1 Reagents ................................................................................. 124
2 ~/ q& M4 ^: y7 h8.2.2 Solutions ................................................................................ 124# p+ N7 n. E6 A3 n, Z6 z& \
8.3 Procedure of Harvesting Cells for Karyotyping+ {1 x9 q+ M1 _7 z
and FISH Analysis ............................................................................. 124
1 {& |  a+ a. ?- x7 EReferences ................................................................................................... 126
, f% J' _4 R# H- ?4 M9 Method for the Derivation of Induced Pluripotent) H" ?) f9 ^0 \1 ]" j; w$ _. @
Stem Cells from Human Hair Follicle Keratinocytes ............................ 127
3 v  z) M! H8 W9.1 Introduction ........................................................................................ 127& Z- u% Z0 v- Q  P! M- r
9.2 Materials ............................................................................................ 129$ Y5 p2 v$ R( t- g1 s9 W0 B
9.2.1 NIH-3T3/293T Cells .............................................................. 129
/ O. x" S7 R0 h& _2 }  N9.2.2 Keratinocyte Derivation from Plucked Hair Follicles ........... 129
  q5 k% z2 v/ i: E2 Q6 y9.3 Methods ............................................................................................. 130
- V: q2 i4 _7 J, e; o# [1 ~0 o( c9.3.1 NIH-3T3 and 293T Culture Methods .................................... 130
8 n7 C& j1 K2 q" u9.3.2 Keratinocyte Culture Methods ............................................... 132: l$ v& h: T5 N  }3 o7 ?, N
9.3.3 Preparation of the STEMCCA Virus for Infection ................ 133
5 m$ g% M: v2 u; S. v7 }3 R* k9.3.4 Derivation of iPSCs from Hair Keratinocytes ....................... 1345 W3 @5 o) Q  ~( F7 j. ]8 M$ T
References ................................................................................................... 137