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Current Protocols in Stem Cell Biology
- E  k! a' t# v; x6 |' GLast Update: July 07, 2009
- ]8 k: Y- M! B# ^' SPrint ISSN: 1941-7322
9 c3 W- ?% e/ q$ ?' AOnline ISSN: 1938-8969( `) T+ J/ [5 x& V
Table of Contents7 {1 [7 G0 {- T/ m' e% Y
Foreword
  h& J: ^! A/ b; h- vPreface
2 M9 t9 |- K! ^4 | Chapter 1 Embryonic and Extraembryonic Stem Cells
# Z% n8 y  I+ t* o" w Section A Isolation of Embryonic Stem Cells & ?8 j( m; l) M. u$ {9 E
Isolation of Embryonic Stem Cells
; P& F- Q1 u2 M7 {3 G, t+ YUNIT 1A.1 Derivation and Characterization of Nonhuman Primate Embryonic Stem Cells + s5 a) h  \5 N: X8 A
UNIT 1A.2 Derivation of hESC from Intact Blastocysts
) k9 ^  R8 }; ~' P5 X% |7 V, r6 m! I: pUNIT 1A.3 Reprogramming Primordial Germ Cells (PGC) to Embryonic Germ (EG) Cells ' E" V2 x/ v% h! K0 d) ]6 x
UNIT 1A.4 Derivation and Propagation of hESC Under a Therapeutic Environment ! H! M( z' N  k( ]$ i
Section B Characterization of Embryonic Stem Cells 1 t* u$ x& z' W: |7 {7 ^
UNIT 1B.1 Proteomic Analysis of Pluripotent Stem Cells ( f! U5 [, t$ K1 h; l$ {
UNIT 1B.2 Gene Expression Analysis of RNA Purified from Embryonic Stem Cells and Embryoid Body–Derived Cells Using a High‐Throughput Microarray Platform
9 ]" G5 t2 p' K$ G  R7 h% NUNIT 1B.3 Phenotypic Analysis of Human Embryonic Stem Cells
. R; a8 B4 P6 i2 D8 f$ @9 gUNIT 1B.4 Isolation of Human Embryonic Stem Cell–Derived Teratomas for the Assessment of Pluripotency
$ H: l# p  U9 d% c) P! V; jUNIT 1B.5 Tandem Affinity Purification of Protein Complexes in Mouse Embryonic Stem Cells Using In Vivo Biotinylation 0 M+ k2 t- m2 P2 m: ^  S7 Z1 w
Section C Culture and Maintenance of Undifferentiated Embryonic Stem Cells - C2 A' ~# ]. Q9 h
Culture and Maintenance of Undifferentiated Embryonic Stem Cells
! ]3 j4 K+ X( cUNIT 1C.1 Expansion of Human Embryonic Stem Cells In Vitro : X1 c1 _; W. v
UNIT 1C.2 Defined, Feeder‐Independent Medium for Human Embryonic Stem Cell Culture
% y) x/ _2 {5 N1 sUNIT 1C.3 Isolation and Propagation of Mouse Embryonic Fibroblasts and Preparation of Mouse Embryonic Feeder Layer Cells
- k! \* j% c$ w3 sUNIT 1C.4 Culture of Mouse Embryonic Stem Cells . B- `: t- y6 Z5 X3 F/ g
UNIT 1C.5 Preparation of Autogenic Human Feeder Cells for Growth of Human Embryonic Stem Cells # t5 S  }8 z3 ]6 ~$ O. l% J9 ~8 C
UNIT 1C.6 Isolation of Human Placental Fibroblasts
% @8 d6 M# r, k7 n) a) m0 @UNIT 1C.7 Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells 7 k) l2 x5 B" S# x
UNIT 1C.8 Cryopreservation of Dissociated Human Embryonic Stem Cells in the Presence of ROCK Inhibitor   I2 U/ e* Y! U1 E5 V" H, S5 V
Section D Germ Layer Induction/Differentiation of Embryonic Stem Cells
6 O( f3 {2 c% H9 Q% ]7 w! eUNIT 1D.1 Germ Layer Induction in ESC—Following the Vertebrate Roadmap
' c2 {1 ^. Q1 }6 J& }( s5 {( EUNIT 1D.2 Formation and Hematopoietic Differentiation of Human Embryoid Bodies by Suspension and Hanging Drop Cultures / @! b! d8 x5 f& L
UNIT 1D.3 Directed Differentiation of Human Embryonic Stem Cells as Spin Embryoid Bodies and a Description of the Hematopoietic Blast Colony Forming Assay - J, L% w4 [, t( {5 i: h
UNIT 1D.4 Differentiation of Human Embryonic Stem Cells in Adherent and in Chemically Defined Culture Conditions # h" A, ^2 ?; I/ H) w. ?. h
UNIT 1D.5 Isolation and Differentiation of Xenopus Animal Cap Cells : N6 D# t7 x6 ?0 ~, R2 B' I: D0 ?  g
Section E Extraembryonic Lineages ! X9 @" n  q7 j+ D
Isolation of Stem Cells from Extraembryonic Tissues . b0 t4 O6 c7 M4 T' \
UNIT 1E.1 Isolation of Human Placenta‐Derived Multipotent Cells and In Vitro Differentiation into Hepatocyte‐Like Cells ) A3 Y. w1 D- r$ W* H" Q  ^
UNIT 1E.2 Isolation of Mesenchymal Stem Cells from Amniotic Fluid and Placenta
2 ?: A4 y( E7 u5 AUNIT 1E.3 Isolation of Amniotic Epithelial Stem Cells & X! l6 o$ O* G% I3 Y% k/ N; `
UNIT 1E.4 Isolation and Manipulation of Mouse Trophoblast Stem Cells
  e% \! X* a0 \7 b5 }( L Section F Mesodermal Lineages
8 T- y# @$ m* V$ @) {UNIT 1F.1 Differentiation of Embryonic Stem Cells into Cartilage Cells
2 I7 Z; c8 a. @* t1 PUNIT 1F.2 Differentiation of Human Embryonic Stem Cells to Cardiomyocytes by Coculture with Endoderm in Serum‐Free Medium
! I& C) d2 O1 L# a* hUNIT 1F.3 Isolation of Hematopoietic Stem Cells from Mouse Embryonic Stem Cells
5 i0 `) z1 F5 Q7 I) QUNIT 1F.4 Differentiation of Mouse Embryonic Stem Cells into Blood
9 c6 d) ~! d4 T, j  ]: ?( k9 y  ^UNIT 1F.5 Endothelial Differentiation of Embryonic Stem Cells
# F. F6 X3 u8 f4 c! mUNIT 1F.6 Hematopoietic Differentiation of Human Embryonic Stem Cells by Cocultivation with Stromal Layers
7 x$ l. G: j0 w1 B5 C* v* _* MUNIT 1F.7 TLX1 (HOX11) Immortalization of Embryonic Stem Cell–Derived and Primary Murine Hematopoietic Progenitors 1 J* A% ^5 l6 Q) n
UNIT 1F.8 Differentiation of Multipotent Mesenchymal Precursors and Skeletal Myoblasts from Human Embryonic Stem Cells
, ~+ Q" m( k3 v' o% h" W( | Section G Endodermal Lineages
- c% |- {9 u7 }9 B% c$ H1 ^( zUNIT 1G.1 The Differentiation of Distal Lung Epithelium from Embryonic Stem Cells 3 j2 {8 t  X3 c4 c6 P, D5 e
UNIT 1G.2 Pancreas Differentiation of Mouse ES Cells ! w0 @" O* [9 ], {
UNIT 1G.3 Differentiation of Embryonic Stem Cells into Anterior Definitive Endoderm - D# c1 S) _( i, |) [! r+ K9 M
Section H Ectodermal Lineages ( L+ l% t' e0 M* K" l
UNIT 1H.1 Differentiation of Mouse Embryonic Stem Cells to Spinal Motor Neurons * }* y4 v( n- C$ ]1 a
Chapter 2 Somatic Stem Cells 6 n' D% L+ c* q
Section A Hematopoietic Stem Cells
" t( U8 W) O* VHematopoietic Stem Cells 5 A! @3 {, u( B7 h7 f
UNIT 2A.1 Isolation of Mononuclear Cells from Human Cord Blood by Ficoll‐Paque Density Gradient
0 R! ~# K- E; p; E0 G3 pUNIT 2A.2 Isolation of Hematopoietic Stem Cells from Human Cord Blood
" C: |- T1 L; H2 M" FUNIT 2A.3 Isolation of Mesenchymal Stem Cells from Human Cord Blood
' S5 y9 _0 u! t+ \& s$ RUNIT 2A.4 Isolation and Assessment of Long‐Term Reconstituting Hematopoietic Stem Cells from Adult Mouse Bone Marrow
* J5 S: h3 }  x( V$ O# UUNIT 2A.5 Analysis of the Hematopoietic Stem Cell Niche
: W% e4 P3 h$ KUNIT 2A.6 Analysis and Manipulation of Hematopoietic Progenitor and Stem Cells from Murine Embryonic Tissues
( ~# j# N6 `  n  RUNIT 2A.7 High Level In Vitro Expansion of Murine Hematopoietic Stem Cells 6 D) I4 ]3 F( V6 U+ e" ~6 X. M! y, r
UNIT 2A.8 Isolation and Visualization of Mouse Placental Hematopoietic Stem Cells 4 s$ T$ A" H" \3 w- R
Section B Non‐Hematopoietic Bone Marrow‐Derived Stem Cells
; P5 s$ [5 m8 A8 mUNIT 2B.1 Isolation and Characterization of Mesoangioblasts from Mouse, Dog, and Human Tissues ) _6 {) k8 d  Q, c4 _7 j+ f! N" C4 u
UNIT 2B.2 Purification and Culture of Human Blood Vessel–Associated Progenitor Cells ; E' u2 v! |( L1 u
UNIT 2B.3 Isolation, Culture, and Differentiation Potential of Mouse Marrow Stromal Cells . {1 H3 w! |* ?1 w1 \
Section C Cardiovascular Stem Cells ( I7 Z4 j0 }9 c3 C% K
UNIT 2C.1 Isolation and Characterization of Endothelial Progenitor Cells from Human Blood
  Z: T; A7 k: }* R6 `UNIT 2C.2 Derivation of Epicardium‐Derived Progenitor Cells (EPDCs) from Adult Epicardium
0 F1 k. r- l( a8 Z1 H6 d Section D Neural Stem Cells 8 s" p7 I: R) u) d) h% `: `) i) o
UNIT 2D.1 Generation of Cultured Oligodendrocyte Progenitor Cells from Rat Neonatal Brains
8 m; L  t8 @/ W4 S' |! V/ Q' l+ c7 KUNIT 2D.2 Isolating, Expanding, and Infecting Human and Rodent Fetal Neural Progenitor Cells " \( S, [6 k- Y
UNIT 2D.3 Long‐Term Multilayer Adherent Network (MAN) Expansion, Maintenance, and Characterization, Chemical and Genetic Manipulation, and Transplantation of Human Fetal Forebrain Neural Stem Cells
" c8 t+ P' U) ]0 ?4 h6 A* B, K Chapter 3 Cancer Stem Cells ' `1 i# z  B& b* _
UNIT 3.1 Colon Cancer Stem Cells * J# R8 s, b' A
UNIT 3.2 In Vivo Evaluation of Leukemic Stem Cells through the Xenotransplantation Model 5 S& d7 u( O/ |) p- b
Chapter 4 Manipulation of Potency
8 e& D" i7 c" v2 R Section A iPS Cells 2 E. |5 |9 z+ H4 d: p
UNIT 4A.1 Human iPS Cell Derivation/Reprogramming
7 h, S0 n# [+ L/ ], T) [UNIT 4A.2 Generation and Characterization of Human Induced Pluripotent Stem Cells
  X) j& j( u& m: Y  R Section B Nuclear Transfer 2 o. ~7 q# h( n: m( d5 U& ~
UNIT 4B.1 Heterokaryon‐Based Reprogramming for Pluripotency
1 ~7 Q$ a  V! ?& q' Y# } Chapter 5 Genetic Manipulation of Stem Cells / ?5 ~+ B( [% q3 V
Section A Lineage Tracers in Stem Cells 7 U) o6 z/ H# w+ c  n9 s
UNIT 5A.1 Imaging Neural Stem Cell Fate in Mouse Model of Glioma
- m+ q6 N2 o  nUNIT 5A.2 Functional Analysis of Adult Stem Cells Using Cre‐Mediated Lineage Tracing 9 H! f  j! @. y" I! l# u: S. M
Appendix 1 Useful Information $ j  W4 F+ i# @0 @& T( ^; W/ J9 A
APPENDIX 1A Guidelines for the Conduct of Human Embryonic Stem Cell Research / _% j) }  L4 U3 ?' ~3 G
APPENDIX 1B ISSCR Guidelines for the Clinical Translation of Stem Cells
4 |9 V1 M+ V: I, J) i Appendix 2 Laboratory Equipment : W2 [/ S' k5 l, Z# D
APPENDIX 2 Standard Laboratory Equipment , A2 T/ p3 x7 d% U
Appendix Suppliers

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4 d2 F5 i9 W3 h+ @/ ^. F, K" N我有UNIT 1D.2 Formation and Hematopoietic Differentiation of Human Embryoid Bodies by Suspension and Hanging Drop Cultures