en-usLUSR2L8UUDifferentiationDifferentiation of Stem Cells/webroot/web/html/lsr/solutions/applications/stem_cell_research<p>The hallmark of stem cells is their ability to self-renew and to differentiate
into different cell types. The induction of stem cell differentiation into a given
cell type often requires a specific combination of media and factors. This section
provides a brief overview of methods for differentiating human embryonic stem cells
(hESCs) into different cell types.</p>
<p><strong>Related Topics:</strong> <a href="/evportal/destination/solutions?catID=LUSR18ESH">Stem
Cell Research</a>, <a href="/evportal/destination/solutions?catID=LUSR1TC4S">Isolation
and Maintenance of Stem Cells</a>, <a href="/evportal/destination/solutions?catID=LUSR3BMNI">Transfection
and Transduction of Stem Cells</a>, <a href="/evportal/destination/solutions?catID=LUSR41KSY">Analysis
of Stem Cells</a>, and <a href="/evportal/destination/solutions?catID=ND1HZL15">Stem
Cells in Therapeutics and Research</a>.</p>
Methods for Stem Cell Differentiation<p>As with culturing stem cells, methods of differentiation depend on the type of
stem cell, the species, target lineages, and somatic cell types. When stem cells are
being induced to differentiate, it is essential that the progress be tracked and that
the phenotype of the cells be confirmed. The lineages and identities of differentiated
cell types can be analyzed using PCR techniques such as <a href="/en-us/category/real-time-pcr-detection-systems">real-time
PCR</a> or <a href="/en-us/category/digital-pcr">digital PCR</a>, <a href="/en-us/product/s3e-cell-sorter">cell
sorting/flow cytometry</a>, immunocytochemistry, <a href="/en-us/category/western-blotting">western
blotting</a>, and <a href="/en-us/category/bio-plex-multiplex-system">biomarker analysis</a>.</p>
<p><img src="/webroot/web/images/lsr/solutions/applications/stem_cells/stem_cell_research/application_detail/sca12_img1.gif"
alt="Differentiation of Embryonic Stem (ES) and induced pluripotent stem (iPS) cells."
width="370" height="731" /></p>
<p class="caption"><strong>ESCs and induced pluripotent stem cells (iPSC) can form
embryoid bodies, which can differentiate into cells of the ectoderm, mesoderm, and
endoderm.</strong></p>
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The Embryoid Body<p>One of the oldest methods for stem cell differentiation is the generation of embryoid
bodies (EBs). Generally, when stem cells are cultured without an adherent surface,
feeder cells, or a complex matrix, the cells aggregate. These aggregated cells spontaneously
differentiate. An EB contains all three germ layers.</p>
<p>EBs are still often used as the initial stage of differentiation for <a href="/en-us/applications-technologies/stem-cell-research#3">embryonic
stem cells (ESCs)</a> and <a href="/en-us/applications-technologies/stem-cell-research#6">induced
pluripotent stem cells (iPSCs)</a>. All downstream differentiated cells are derived
from this initial structure. There are several methods for forming EBs, including
suspension culture, hanging-drop culture, and culture in semisolid media. All protocols
start with detaching a high-density cell culture from the dish, either enzymatically
or with versine, depending on the properties of the cells.</p>
<p><strong>Suspension culture</strong> is the most common method for EB formation
but also the hardest to control, for both EB size and shape; EBs can become large
and irregularly shaped in suspension. Suspension culture is scalable to bioreactors,
although the exact methods and factors such as stir rate that affect the physical
dimensions of EBs must be determined empirically.</p>
<p><strong>Hanging-drop</strong> culture gives smaller and much more uniform EBs.
The size of the EBs can be controlled by controlling the number of cells in each drop.
Making the drops (usually ~20 μl) was formally labor intensive, and hence the numbers
of EBs generated was low. Now, higher-throughput hanging-drop methods using arrays
have been developed and used for anticancer-drug sensitivity testing (Hsaio et al.
2012).</p>
<p><strong>Semisolid media</strong>, usually methylcellulose, can be used to suspend
the EBs. This method is efficient for making EBs, but is not as scalable as suspension
culture.</p>
<p>ESCs form EBs in about four days, and are often allowed to grow for weeks. All
three germ layers are present, but the composition of the media influences the ratios
of cell types and lineages.</p>
<p><img src="/webroot/web/images/lsr/solutions/applications/stem_cells/stem_cell_research/application_detail/sca12_img2.jpg"
alt="" width="257" height="198" /></p>
<p class="caption"><strong>Embryoid bodies after growing in suspension for eight days.</strong>
Image courtesy of Dr. Miguel Esteban.</p>
<p>Depending on the desired end point, the presence of all three germ layers in EBs
can be a disadvantage. When a specific lineage or cell type is required, cultures
must be depleted of the unwanted cell types. Therefore, many protocols have been developed
for directly differentiating ESCs and iPSCs without using EBs. The methods are specific
for the species, lineage, and cell type.</p>
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Cells of the Ectoderm<p>The central nervous system, hair, and the epidermis are all derived from the ectoderm.
There are several protocols for producing neural progenitor cells from undifferentiated
cultures. One of these protocols is described below (Zhang et al. 2001). This protocol
has been the basis for the generation of a number of different neuronal cell types.</p>
<p>To induce EBs to form neurons, the culture medium is replaced with neural basal
media containing bFGF (basic fibroblast growth factor) heparin, and N2 supplement.
N2 supplement consists of transferrin, insulin, progesterone, putrescine, and selenite.
Two days later, attachment of the differentiating EBs is induced by plating them onto
dishes coated with laminin or polyornithine. After an additional 10–11 days
in culture, the EBs differentiate into primitive neuroepithelial cells. The identity
of the cells can be confirmed by staining for PAX6 (paired box protein 6, a transcription
factor), SOX2 (sex-determining region Y-box 2, another transcription factor), and
N-cadherin (a calcium-dependent cell adhesion molecule specific to neural tissue).</p>
<p>At this point, it is possible to differentiate the neuroepithelial cells into specific
cell types of the central nervous system including motor neurons (Li et al. 2005),
dopaminergic neurons (Yan et al. 2005), and oligodendrocytes (Nistor et al. 2005).</p>
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Cells of the Mesoderm<p>Cells of the mesoderm form most of the body's internal supporting structures, including
blood, muscle, bone, cartilage, and heart. Because new mesodermal cells have potential
for use in the treatment of common ailments such as osteoarthritis, osteoporosis,
and cardiovascular disease, there has been a major focus on protocols for stem cell
differentiation into mesodermal cells.</p>
<p>Cardiomyocytes develop spontaneously from 10-day-old EBs plated onto gelatin-coated
plates (Kehat et al. 2001). Fortunately, although they are a small percentage of the
cells, cardiomyocytes are easy to identify due to their hallmark rhythmic contractions.
Cardiomyocytes can be separated from the rest of the differentiating culture by <a
href="/en-us/product/s3e-cell-sorter">cell sorting</a> using antibodies against cardiac
markers (Xu et al. 2002).</p>
<p>An alternative method for deriving cardiomyocytes is to transfect a stem cell culture
with a viral vector containing a drug-resistance gene driven by the alpha-myosin heavy
chain promoter. Subsequent selection for drug resistance enables the selection of
cells that differentiate into cardiomyocytes (Zhao and Lever 2007).</p>
<p>The differentiation of stem cells into cells within the hematopoietic lineage has
long been of important clinical interest for cancers of the blood, such as leukemia.
Early work led to the development of techniques for differentiating human embryonic
stem cells (hESCs) cells into most of the cells of the hematopoietic system (Keller
et al. 1993, Kaufman et al. 2001). More recently , the potential for creating replacement
cells for blood transfusion, blood cell disease, vascular disease (with endothelial
cells), and immunodeficiency disorders has increased interest in the development of
techniques for differentiating both iPSCs and hESC into cells of the hematopoietic
and vascular systems, and clinical trials have been undertaken for stem cell-derived
therapies for leukemia, lymphoma, and sickle cell disease (Nature Biotechnology News
2014).</p>
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Cells of the Endoderm<p>The endoderm forms many of the internal organs, including the pancreas and the
liver. High rates of diabetes and liver disease have made the production of insulin-secreting
cells and hepatocytes key goals in the field of stem cell research.</p>
<p>Type 1 diabetes is caused by the destruction of the insulin-secreting beta cells
of the Islet of Langerhans in the pancreas. Current treatment options include pancreatic
transplants or the infusion of donor beta cells. However, donors are in short supply,
and beta cell transplantation is usually not a permanent cure due to immune response
in the recipient, leading to destruction of the donated beta cells.</p>
<p>It is now possible to make human embryonic stem cells into all pancreatic cell
lineages (Guo and Hebrok 2009). However, the beta-like cells produced during the complex
differentiation process are not efficient insulin producers and are not as completely
responsive to cell signaling as native beta cells (Furth and Atala 2009). A breakthrough
in this line of research was recently reported in which large numbers of functional
human pancreatic β cells were generated in vitro, providing an unprecedented
cell source for drug discovery and cell transplantation therapy in diabetes (Pagliuca
et al. 2014). Progress toward developing liver cells (hepatocytes) for transplantation
has been slow. Stem cells have been differentiated into hepatocyte-like cells using
several methods (Hay et al. 2008, Basma et al. 2009, Szkolnika et al. 2014) but are
generally not suitable for transplantation.</p>
<p><img src="/webroot/web/images/lsr/solutions/applications/stem_cells/stem_cell_research/application_detail/sca12_img3.jpg"
alt="" width="534" height="721" /></p>
<p class="caption"><strong>Teratomas composed of tissues derived from the three germ
layers produced after injection of an ESC cell line into immunosuppressed mice.</strong>
Image courtesy of Dr. Miguel Esteban.</p>
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Conclusion<p>The ability of stem cells to differentiate into nearly any cell in the body gives
them the potential to form the basis of therapies for many conditions. As research
moves forward, standardized techniques for stem cell culture and differentiation will
be developed. These new techniques will lay the foundation for research into cells
and tissues and future stem cell therapies.</p>
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References<p>Basma H et al. (2009). Differentiation and transplantation of human embryonic stem
cell-derived hepatocytes. Gastroenterology 136, 990–999. PMID: <a class="new-target-image"
href="http://www.ncbi.nlm.nih.gov/pubmed/19026649" target="_blank" rel="noopener noreferrer">19026649
</a></p>
<p>Furth ME and Atala A (2009). Stem cell sources to treat diabetes. J Cell Biochem
106, 507–511. PMID: <a class="new-target-image" href="http://www.ncbi.nlm.nih.gov/pubmed/19130494"
target="_blank" rel="noopener noreferrer">19130494</a></p>
<p>Guo T and Hebrok M (2009). Stem cells to pancreatic beta-cells: New sources for
diabetes cell therapy. Endocr Rev 30, 214–227. PMID: <a class="new-target-image"
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<p>Hay DC et al. (2008). Efficient differentiation of hepatocytes from human embryonic
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<p>Hsaio AY et al. (2012). 384 hanging drop arrays give excellent Z-factors and allow
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<p>Kaufman DS et al. (2001). Hematopoietic colony-forming cells derived from human
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<p>Kehat I et al. (2001). Human embryonic stem cells can differentiate into myocytes
with structural and functional properties of cardiomyocytes. J Clin Invest 108, 407–414.
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<p>Keller G et al. (1993). Hematopoietic commitment during embryonic stem cell differentiation
in culture. Mol Cell Biol 13, 473–486. PMID: <a class="new-target-image" href="http://www.ncbi.nlm.nih.gov/pubmed/8417345"
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<p>Li XJ et al. (2005). Specification of motor neurons from human embryonic stem cells.
Nat Biotechnol 23, 215–221. PMID: <a class="new-target-image" href="http://www.ncbi.nlm.nih.gov/pubmed/15685164"
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<p>Nature Biotechnology News (2014). Novartis wades into stem cell therapies. Nat
Biotech 32, 969. <a class="new-target-image" href="http://doi.org/doi:10.1038/nbt1014-969e"
target="_blank" rel="noopener noreferrer">doi:10.1038/nbt1014-969e</a></p>
<p>Nistor GI et al. (2005). Human embryonic stem cells differentiate into oligodendrocytes
in high purity and myelinate after spinal cord transplantation. Glia 49, 385–396.
PMID: <a class="new-target-image" href="http://www.ncbi.nlm.nih.gov/pubmed/15538751"
target="_blank" rel="noopener noreferrer">15538751</a></p>
<p>Pagliuca FW et al. (2014). Generation of Functional Human Pancreatic β Cells
In Vitro. Cell 159, 428–439. PMID: <a class="new-target-image" href="http://www.ncbi.nlm.nih.gov/pubmed/25303535"
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<p>Yan Y et al. (2005). Directed differentiation of dopaminergic neuronal subtypes
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<p>Zhao J and Lever AM (2007). Lentivirus-mediated gene expression. Methods Mol Biol
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<p>Zhang SC et al. (2001). In vitro differentiation of transplantable neural precursors
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Karen MossDifferentiation of Stem Cells12/27/11 11:43 AM12/27/21 11:59 AMAE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA,VNenLSR/LSR/Applications/Stem_Cell_ResearchN0Stem Cell Research
/en-us/applications-technologies/differentiation-stem-cells?ID=LUSR18ESH
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