Multi-ponent differentiation of mesenchymal stem cells

Mesenchymal Differentiation Standard published protocols were used to promote differentiation of passage 3-6 MSCs along mesenchymal lineages, including osteogenic [2], adipogenic [1], and chondrogenic lineages [3,4].

Briefly, to induce osteogenic differentiation, confluent MSCs were incubated in osteogenic medium (control medium, supplemented with 10 nM dexamethasone, 50 ug/ml L-ascorbic acid phosphate, and 5 mM beta-glycerophosphate [all from Sigma-Aldrich]) for 12 days, before being stained for alkaline phosphatase activity and mineralization using the von Kossa stain. Adipogenic differentiation was stimulated by culturing confluent MSCs in adipogenic medium (control medium with 10 nM dexamethasone, 50 ug/ml L-ascorbic acid phosphate, 500 uM isobutylmethyxanthine [IBMX; Sigma-Aldrich] and 60 uM indomethacin [Sigma-Aldrich]) for 12 days, before identifying adipocytes by oil red O staining. MSCs were grown in micromass cultures to induce chondrogenic differentiation. 2 x105 cells in chondrogenic medium (DMEM containing 110 ug/ml sodium pyruvate, 100 U/ml penicillin, 100 ug/ml streptomycin, 1% human recombinant insulin, human transferrin, and selenous acid [ITS] + Premix [BD Biosciences, Oxford, U.K., http://www.bd.com], 50 ug/ml L-ascorbic acid phosphate, 100 nM dexamethasone, 50 ug/ml proline [Sigma-Aldrich], and 10 ng/ml recombinant human transforming growth factor?beta3 [TGF-beta3; R&D Systems, Oxon, UK, http://www.rndsystems.com]) were placed in universals and pelleted by centrifugation at 800 rpm for 5 minutes, before incubation for 15 days.



References:
1. Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284:143?147.
2. Jaiswal N, Haynesworth SE, Caplan AI et al. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 1997;64:295?312.
3. Yoo JU, Barthel TS, Nishimura K et al. The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells. J Bone Joint Surg Am 1998;80:1745?1757.
4. Mackay AM, Beck SC, Murphy JM et al. Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow. Tissue Eng 1998;4:415?428.

Stem Cell Research Methods

Developmental Biology/Embryonic Stem Cell Protocols
Protocol Online logo ... Top : Developmental Biology : Embryonic Stem Cell ... 1999-2006 Protocol Online, All rights reserved. ...www.protocol-online.org/prot/Developmental_Biology/Embryonic_Stem_Cell/
BioProtocols--Protocol online, Biological Information, Biomedical ...
There are hundreds of protocols in the areas of molecular biology, cellular biology, metabolism, oncology, neurology, stem cell research, animal experiments ...www.lab-manual.com/
Stem Cell Harvest Protocol for National Human Neural Stem Cell ...
Tissue Protocol. Stem Cell Protocol ... Prepare remainder of fresh brain and other tissues according to standard protocol, below. ...www.nhnscr.org/assist/sc_protocol.htm
Rat Cortical Stem Cell Culture Protocol
Remove the cryovial containing frozen rat cortical stem cells from the liquid nitrogen. Using a 2 mL pipette, immediately add 1 mL of fresh pre-warmed media ...www.rndsystems.com/stem_cell_protocol_detail_objectname_RatCortical.aspx Protocols- Cell Biology and Cell Culture > ES Stem Cell Biology ...
Embryonic Stem Cell Protocol. tips and protocol for culture. Allan Bradley Lab, Baylor College of Medicine, Houston, Texas - [Read more Embryonic Stem Cell ...www.molecularstation.com/protocol-links/Cell-Biology-Cell-Culture/ES_Stem_Cell_Biology_and_Culture/
Thomson Lab Stem Cell Protocols
stem cell colony. Media and reagents; Thawing ES Cells; Splitting ES Cells on MEF's (stem ... Human Embryonic Stem (ES) Cell Protocols. Media and Reagents ...ink.primate.wisc.edu/~thomson/protocol.html
Culture of Human Embryonic Stem Cells (hESC) [Stem Cell Information]
The National Institutes of Health resource for stem cell research ... supplier's protocols but we are adapting them to one simple protocol outlined below: ...stemcells.nih.gov/research/NIHresearch/scunit/culture.asp
ISSCR :: Stem Cell Science : Stem Cell Books
Furthermore, chapters detailing adult stem cell plasticity should be read in .... Each fully tested protocol is described in step-by-step detail and ...www.isscr.org/science/books.htm - 62k
Murine Embryonic Stem Cell Protocol:. 129 ES Cells. Adapted from Wesselschmidt, RL Primogenix, Inc. robin@primogenix.com. Table 1: Preparation of 250 mL 129 ...www.hyclone.com/advancestem/PDF/Murine%20ESC%20129%20ES%20PS%2000006.
Protocols- Stem Cell Protocols > Feeder Cell Preparation Protocols
The ES culture medium is supplemented with recombinant leukemia inhibitory factor (LIF) to help maintain the cells as pluripotent stem cells. This protocol ...www.molecularstation.com/protocol-links/stem-cell-protocols/feeder-cell-preparation-protocols/
Tissue Harvest Protocol for the National Human Neural Stem Cell ...
Assist Donors ................................... Information for Donors · Consent Forms. Tissue Protocol. Stem Cell Protocol ...www.nhnscr.org/assist/tissue_protocol.htm
Hoechst 33342 HSC Staining and Stem Cell Purification Protocol
HO protocol. 1. Goodell, M.A. 8/99. Hoechst 33342 HSC Staining and Stem Cell Purification Protocol. (see Goodell, M., et al. (1996) J Exp Med 183, 1797-806) ...www.bcm.edu/labs/goodell/protocols/goodell_hoechst.pdf
protocol
BMT CTN Protocol 0102 -- A Trial of Tandem Autologous Stem Cell Transplants +/- Post Second Autologous Transplant Maintenance Therapy versus Single ...https://web.emmes.com/study/bmt/protocol/protocol.html
ESCRO - Embryonic Stem Cells: Research Compliance, University of ...
ESCRO has the final sign-off on stem cell protocol approvals. Final ESCRO approval will not be given until the ESCRO receives documentation of all required ...escro.uconn.edu/investigators.html
Invitrogen Online Ordering: Lipofectamine™ 2000 Transfection ...
Lipofectamine™ 2000 Transfection Protocol for Human Mesenchymal Stem Cells ... (or siRNA) into human MSC mesenchymal stem cells (Cambrex BioScience, Cat. ...https:/.../index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeId=3E32DAE7CD11EA19E3BA69301D2079BA - Commentary: A Novel Protocol That Allows Short-Term Stem Cell ...
novel protocol that allows short-term stem cell expan-. sion of both committed and pluripotent hematopoietic. progenitor cells suitable for clinical use. ...linkinghub.elsevier.com/retrieve/pii/S1079979601800024
Bone Marrow Transplantation - Non-TBI stem cell transplantation ...
Four patients have undergone stem cell transplantation following this new protocol, two (P1-SIB1 and P2-SIB2) with HLA-compatible sibling donors and two ...www.nature.com/bmt/journal/v32/n7/full/1704219a.html

Milestones of Stem Cell Research

(from Wikipedia,the free encyclopedia)
1960s - Joseph Altman and Gopal Das present scientific evidence of adult neurogenesis, ongoing stem cell activity in the brain; their reports contradict Cajal's "no new neurons" dogma and are largely ignored.
1963 - McCulloch and Till illustrate the presence of self-renewing cells in mouse bone marrow.
1968 - Bone marrow transplant between two siblings successfully treats SCID.
1978 - Haematopoietic stem cells are discovered in human cord blood.
1981 - Mouse embryonic stem cells are derived from the inner cell mass by scientists Martin Evans, Matthew Kaufman, and Gail R. Martin. Gail Martin is attributed for coining the term "Embryonic Stem Cell".
1992 - Neural stem cells are cultured in vitro as neurospheres.
1997 - Leukemia is shown to originate from a haematopoietic stem cell, the first direct evidence for cancer stem cells.
1998 - James Thomson and coworkers derive the first human embryonic stem cell line at the University of Wisconsin-Madison.
2000s - Several reports of adult stem cell plasticity are published.
2001 - Scientists at Advanced Cell Technology clone first early (four- to six-cell stage) human embryos for the purpose of generating embryonic stem cells.[30]
2003 - Dr. Songtao Shi of NIH discovers new source of adult stem cells in children's primary teeth.[31]
2004-2005 - Korean researcher Hwang Woo-Suk claims to have created several human embryonic stem cell lines from unfertilised human oocytes. The lines were later shown to be fabricated.
2005 - Researchers at Kingston University in England claim to have discovered a third category of stem cell, dubbed cord-blood-derived embryonic-like stem cells (CBEs), derived from umbilical cord blood. The group claims these cells are able to differentiate into more types of tissue than adult stem cells.
August 2006 - Rat Induced pluripotent stem cells: the journal Cell publishes Kazutoshi Takahashi and Shinya Yamanaka, "Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors".
October 2006 - Scientists in England create the first ever artificial liver cells using umbilical cord blood stem cells.[32][33]
January 2007 - Scientists at Wake Forest University led by Dr. Anthony Atala and Harvard University report discovery of a new type of stem cell in amniotic fluid.[5] This may potentially provide an alternative to embryonic stem cells for use in research and therapy.[34]
June 2007 - Research reported by three different groups shows that normal skin cells can be reprogrammed to an embryonic state in mice.[35] In the same month, scientist Shoukhrat Mitalipov reports the first successful creation of a primate stem cell line through somatic cell nuclear transfer[36]
October 2007 - Mario Capecchi, Martin Evans, and Oliver Smithies win the 2007 Nobel Prize for Physiology or Medicine for their work on embryonic stem cells from mice using gene targeting strategies producing genetically engineered mice (known as knockout mice) for gene research.[37]
November 2007 - Human Induced pluripotent stem cells: Two similar papers released by their respective journals prior to formal publication: in Cell by Kazutoshi Takahashi and Shinya Yamanaka, "Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors", and in Science by Junying Yu, et al., from the research group of James Thomson, "Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells": pluripotent stem cells generated from mature human fibroblasts. It is possible now to produce a stem cell from almost any other human cell instead of using embryos as needed previously, albeit the risk of tumorigenesis due to c-myc and retroviral gene transfer remains to be determined.
January 2008 - Human embryonic stem cell lines were generated without destruction of the embryo[38]
January 2008 - Development of human cloned blastocysts following somatic cell nuclear transfer with adult fibroblasts[39]

Stem cell funding & policy debate in the US

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1995 - U.S. President Bill Clinton signs into law the Dickey Amendment which prohibited federally appropriated funds to be used for research where human embryos would be either created or destroyed.
02 November, 2004 - California voters approve Proposition 71, which provides $3 billion in state funds over ten years to human embryonic stem cell research.
2001-2006 - U.S. President George W. Bush endorses the Congress in providing federal funding for embryonic stem cell research of approximately $100 million as well as $250 million for research on adult and animal stem cells. He also enacts laws that restrict federally-funded stem cell research on embryonic stem cells to the already derived cell lines.
5 May, 2006 - Senator Rick Santorum introduces bill number S. 2754, or the Alternative Pluripotent Stem Cell Therapies Enhancement Act, into the U.S. Senate.
18 July, 2006 - The U.S. Senate passes the Stem Cell Research Enhancement Act H.R. 810 and votes down Senator Santorum's S. 2754.
19 July, 2006 - President George W. Bush vetoes H.R. 810 (Stem Cell Research Enhancement Act), a bill that would have reversed the Clinton-era law which made it illegal for federal money to be used for research where stem cells are derived from the destruction of an embryo.
07 November, 2006 - The people of the U.S. state of Missouri passed Amendment 2, which allows usage of any stem cell research and therapy allowed under federal law, but prohibits human reproductive cloning.[40]
16 February, 2007 – The California Institute for Regenerative Medicine became the biggest financial backer of human embryonic stem cell research in the United States when they awarded nearly $45 million in research grants.[41]

See also
The American Society for Cell Biology
California Institute for Regenerative Medicine
Genetics Policy Institute
Cancer stem cells
Induced Pluripotent Stem Cell (iPS Cell)