Technique / Cell Biology / Cell culture / ES stem cell culture

Neural differentiation of human ES cells.
(Curr Protoc Cell Biol. 2007 Sep;Chapter 23:Unit 23.7.)
A Cohen M, Itsykson P, E Reubinoff B.

Hadassah University Medical Center, Ein-Kerem, Jerusalem, Israel.

Human embryonic stem cells (hESCs) may be converted into highly enriched cultures of neural precursors under defined culture conditions. The neural precursors can proliferate in culture for prolonged periods of time, and can differentiate in vitro into mature neurons, astrocytes, and oligodendrocytes. The neurons are
functional and have normal electrophysiological properties. After transplantation to the developing rodent brain, the neural precursors migrate extensively into the host brain parenchyma, respond to host brain signals, and differentiate in a
region-specific manner to progeny of the three neural lineages. The establishment
of neuroectodermal precursors from hESCs allows the study of human neurogenesis
in vitro and is an aid in drug discovery. In addition, the neural precursors may potentially serve as a platform for the development of specific functional neural cells for transplantation and gene therapy of neurological disorders. In this
unit, we introduce methods for the derivation, propagation and characterization of hESC-derived neural precursors.

Differentiation of mouse embryonic stem cells and of human adult stem cells into
(Curr Protoc Cell Biol. 2007 Mar;Chapter 23:Unit 23.4.)
Wdziekonski B, Villageois P, Dani C.

CNRS Université de Nice Sophia Antipolis, Nice, France.

The authors describe protocols for culture conditions in which mouse ES cells can be maintained in an undifferentiated state or committed to undergo adipocyte differentiation at a high rate and in a highly reproducible fashion. There is also a protocol for maintaining and differentiating human adult stem cells, isolated form adipose tissue and from bone marrow, into adipocytes. These culture systems provide a powerful means for studying the first step of adipose cell
development and a means to investigate effects of drugs on the biology of
adipocytes. There are also protocols for detection of adipocytes and analysis of
their gene expression.

Maintenance and in vitro differentiation of mouse embryonic stem cells to form
(Curr Protoc Cell Biol. 2007 Mar;Chapter 23:Unit 23.3.)
Kappas NC, Bautch VL.

The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina,
USA.

Embryonic stem (ES) cells, which are derived from developing mouse blastocysts, have the capacity to give rise to all cell types in the adult body. The ability of ES cells to do so has opened the door for novel experimental approaches in the
field of developmental biology. Under appropriate culture conditions, ES cells
will differentiate and form embryoid bodies (EBs). Upon attachment to a
permissive surface, EBs continue a programmed differentiation, and many of the cells differentiated from the EBs reflect those found in the developing embryo and yolk sac, such as hematopoietic cells, endoderm, and endothelial cells. Endothelial cells that arise during ES cell differentiation have the potential to
form primitive blood vessels, comparable to the vessels that first form in vivo.
This unit describes protocols for maintaining ES cells and the subsequent
differentiation of EBs. This unit also provides methods for analyzing vascular
marker expression in differentiated ES cultures.

Mouse embryonic stem cell derivation, and mouse and human embryonic stem cell
(Curr Protoc Cell Biol. 2005 Oct;Chapter 23:Unit 23.2.)
Conley BJ, Denham M, Gulluyan L, Olsson F, Cole TJ, Mollard R.

Monash University, Melbourne, Australia.

Embryonic stem (ES) cells are pluripotent cells derived from developing mouse blastocysts in vitro that maintain long-term self renewal and the capacity to give rise to all cell types in the adult body (including some extraembryonic cell types) when subjected to the appropriate conditions. It is envisaged that the development of methods enabling controlled differentiation of mouse ES cell
counterparts from human blastocysts would enable the provision of an unlimited supply of tissue for cell and tissue transplantation therapies for the repair and replacement of diseased, injured, and senescent tissue. Furthermore, derivation of mouse ES cells has allowed for the generation of thousands of gene-targeted mouse mutants. Culture of mouse ES cells as embryoid bodies (EBs) has provided a
convenient system for studying early mouse developmental processes, including
several aspects of extraembryonic lineage and axis formation associated with the pre- and peri-gastrulating mouse embryo. Relatively little is known regarding the corresponding development of the early human embryo due to limitations associated with the acquisition of relevant tissue material for study. The transfer of methods such as EB formation to human systems should, by association, facilitate a more advanced understanding of similar processes associated with early human
development. This unit describes protocols for isolating mouse embryonic stem cells and methods for propagating, freezing, and producing EBs from both mouse and human embryonic stem cells.

Stem cells: an overview.
(Curr Protoc Cell Biol. 2005 Oct;Chapter 23:Unit 23.1.)
Denham M, Conley B, Olsson F, Cole TJ, Mollard R.

Monash University, Melbourne, Australia.

Stem cells are specialized cells that possess a capacity to undergo self-renewal while at the same time having the ability to give rise to at least one or more differentiated or mature cell type. They therefore represent a fundamental cornerstone during the life of all vertebrates, playing central roles in the production of new and replacement cells for tissues during development and homeostasis, including repair following disease or injury. This unit is a review of stem cells, their roles in development, and their potentials as therapeutic agents.

Induction of ES cell-derived cartilage formation.
(Curr Protoc Cell Biol. 2007 Mar;Chapter 23:Unit 23.5.)
Kramer J, Schlenke P, Rohwedel J.

University of Lübeck, Lübeck, Germany.

This unit describes the protocols used for cultivation of murine embryonic stem
(ES) cells and their differentiation into chondrogenic cell types in vitro. ES
cells cultivated as cellular aggregates, so-called embryoid bodies (EBs),
differentiate spontaneously into chondrogenic cell types recapitulating cellular events of chondro- and osteogenesis. The undifferentiated ES cells differentiate into mesenchymal prechondrogenic cells in the EB outgrowths. These progenitor
cells aggregate and form mesenchymal condensations. During further cultivation, these cells form cartilage nodules, show a phenotype typical for chondroblasts, and start to express marker molecules of cartilage tissue. Later, the chondrocytes become hypertrophic, and finally, marker molecules indicating bone formation can be detected in the nodules. This unit also contains protocols for characterization of the differentiated cells by immunostaining, mRNA-in situ
hybridization, electron microscopy, and RT-PCR analysis.

(Stem cell culture methods reviews)
1: Findikli N, Candan NZ, Kahraman S.
Human embryonic stem cell culture: current limitations and novel strategies.
Reprod Biomed Online. 2006 Oct;13(4):581-90.

2: Emerson SG, Conrad PD.
Clinical application of hematopoietic stem cell culture and expansion.
Cancer Treat Res. 1999;101:377-88.

3: Audet J, Zandstra PW, Eaves CJ, Piret JM.
Advances in hematopoietic stem cell culture.
Curr Opin Biotechnol. 1998 Apr;9(2):146-51.

4: Baribault H, Kemler R.
Embryonic stem cell culture and gene targeting in transgenic mice.
Mol Biol Med. 1989 Dec;6(6):481-92.

5: Young JC, Varma A, DiGiusto D, Backer MP.
Retention of quiescent hematopoietic cells with high proliferative potential
during ex vivo stem cell culture.
Blood. 1996 Jan 15;87(2):545-56.

6: Meuleman N, Tondreau T, Bron D, Lagneaux L.
Human marrow mesenchymal stem cell culture: serum-free medium allows better
expansion than classical alpha-minimal essential medium (MEM).
Eur J Haematol. 2007 Feb;78(2):168.

7: Wang Y, Huso DL, Harrington J, Kellner J, Jeong DK, Turney J, McNiece IK.
Outgrowth of a transformed cell population derived from normal human BM
mesenchymal stem cell culture.
Cytotherapy. 2005;7(6):509-19.

8: Murashov AK, Pak ES, Katwa LC.
Parallel development of cardiomyocytes and neurons in embryonic stem cell
culture.
Biochem Biophys Res Commun. 2005 Jul 8;332(3):653-6.

9: Stem cell culture shock.
Nat Methods. 2005 Mar;2(3):153.

10: Pera MF.
Stem cell culture, one step at a time.
Nat Methods. 2005 Mar;2(3):164-5.

11: Heng BC, Liu H, Cao T.
Feeder cell density--a key parameter in human embryonic stem cell culture.
In Vitro Cell Dev Biol Anim. 2004 Sep-Oct;40(8-9):255-7.

12: Dove A.
Ex vivo stem cell culture
Nat Biotechnol. 1999 Aug;17(8):738.