Stemness: Definition

Definition: Have the capacity of renewal, as well as the generation of diff cells, in other words, have the ability
to generate identical daughter cells as well as to produce daughter cells w/ more restricted potential. The
important criteria about renewal is lifetime renewal capacity, to distinguish them from e.g. transient adult
progenitor cells. Potency should also be elaborated; is the cell capable of generating only one cell type (unipotent),
or is it capable of producing multiple cell types (multipotent)?

Self-Renewal: Defined as the stem cells’ enhanced capacity to replicate in vitro in comparison to “normal”
somatic cells, most notably the ES and ANS (adult neural stem) cells. Serial transfer from host to host can also be
used to asses this capacity, notably for adult HSCs (hematopoietic stem cells). Adult stem cell renewal is best
defined in vivo, demonstrating their lifetime renewal capacity.

Clonality: Defined as the stem cell’s ability to generate progeny quite similar to itself, thus maintaining its
lineage.

Potency: Used to explain how many different types of cells the stem cell can generate. Multipotent stem cells
are definitely stem cells, while unipotent cells are sometimes defined to be stem cell progeny w/ more restricted
self-renewal and potency.

Conclusion: A working definition of a stem cell is a clonal, self-renewing entity that is multipotent and thus can
generate several differentiated cell types

Origin of ES cells: They appear to be the in vitro equivalent of epiblast cells. In the cystic blastocyst stage they
comprise the inner cell mass, later producing the epiblast and all the other adult tissues. No totipotent cell has
been isolated from the early embryo. The embryo cells w/ the highest potency are capable of producing all adult
cell layers, but not the trophectoderm and extraembryonic endoderm & mesoderm. Trophectoderm cells have at
maximum the potency of generating the trophectoderm lineage.

Origin of HSCs: The first site of hematopoiesis is the extraembryonic yolk sac, soon followed by the
intraembryonic aorta-gonad-mesonephros (AGM) region. The AGM is proposed to generate the adult
hematopoietic system and HSCs.

Origin of NSCs: Their development begins w/ formation of the nervous tissue (i.e. neural plate) after
gastrulation, from the ectoderm.

Molecular Characteristics of Stem Cells: First, they appear to express a broad range of receptors and
down-stream signaling components, for pathways such as TGF, Notch, Wnt, and Jak/Stat. Second, they express
many components of regulating their specialized cell-cycle, either arresting at G1 (for quiescent adult stem cells)
or progression through the cell cycle checkpoints promoting rapid cycling (for ES cells and mobilized adult stem
cells). Third, they have elaborate telomere maintenance systems and elevated telomerase activity. Fourth, they
have an extensively remodeled chromatin, acted upon by DNA methylases or transcriptional repressors of HDACs.
Fifth, they have a characteristic posttranscriptional regulatory machinery regulated by RNA helicases. Finally, they
share a high resistance to stress, e.g. multidrug transporters, folding machinery, ubiquitin, and detoxifier systems.
Embryonic Carcinoma (EC) Cells in ES Cell Discovery: Teratocarcinoma cells, when transplanted on
adult tissues, were able to generate similar tumors w/ multiple cell layers. When injected into early embryos, they
participated in normal development, mostly. Some caused tumors, and they generally contributed in modest,
patchy proportions. However, if cultured on feeder cells, they acted exactly like ES cells, w/ much more division
capacity. These stem cells, w/ the much desired characteristics of original EC cells and few of their shortcomings
came to be called embryonic stem (ES) cells. Their hallmark ability is colonizing the germ-line after transplantation.

ES Cells Potency: At least in mouse, they have been shown to be capable of generating entire offspring or at
least all types of fetal cells, thus being regarded as totipotent; however, this is misleading. Totipotent cells are
defined by their ability to generate an entire conceptus, and thus offspring, unaided. Apart from the egg, the only
cells shown to be compatible w/ this definition are cells from early cleavage stages. The murine ES cell are shown
to be unable to generate all the cells of a conceptus. Upon injection, they’re only capable of generating epiblast
or fetal precursor cell lineages. They have never been able to convincingly generate trophectoderm lineage cells.
Therefore, the pluripotent term is coined to indicate their intermediate level of potency, between totipotent cells
and multipotent cells (all intraembryonic cells, but not extraembryonic layers). The general usage of the term
totipotent for ES cell is for reference to their capacity of generating offspring during reproductive cloning.

ES Cell Definition: Pluripotent cells derived from pre- or peri-implantation conceptuses that can form
functional gametes, as well all three somatic cell layers. Another feature is the ability to colonize both the germline
and the somatic layers. Other ES-like cells have been derived from other species mainly from the blastocyst, but
their definition is controversial, regarding different criteria.

Direct Reprogramming of Somatic Cells to a Pluripotent State

Reprogramming: Converting the epigenetic state of a differentiated somatic cell into a pluripotent state.

SCNT: transfer of a somatic nucleus into a human donor oocyte to generate autologous pluripotent cells, also
w/ application in organism cloning. Not used for obtaining human pluripotent cells, because of lack of knowledge
of the underlying procedure and ethical concerns; simply not possible.

Epigenetic Reprogramming: Simultaneous retroviral expression of the four TFs Oct4, Sox2, Klf4, and C-
myc could directly reprogram mouse fibroblasts to pluripotent ES cell-like cells (iPSCs)

IPSC properties: Expression levels of ES cell-specific TFs such as Nanog and endogenous Oct4 and Sox2
comparable to ES cells. In fact, IPSCs reactivate the autoregulatory pluripotency-related transcription network.
This autoregulatory loop is proposed to be the key module for self-maintenance of an ES cell state.

Chromatin immunoprecipitaion and methylation analysis have revealed that the IPSCs have an epigenetic state
similar to that of ES cells. Also capable of teratoma formation and generation of the three germ layers.

Direct Reprogramming Stochasticity: The small percent of the reprogrammed cells in each trial shows
that direct reprogramming is a stochastic process, partially due to heterogeneity of the viral integrations of the
reprogramming factors leading to variegation effects and differential factor expression due to viral silencing.
The Two Methods of Generating Human IPSCs: the first is the conventional method of reprogramming
mouse cells using the same factors (Klf4, Sox2, C-myc and Oct4). The second one employs overexpression of Lin28
and Nanog in addition to Oct4 and Sox2. Anyway, a big problem is the formation of tumors in IPSC-generated
chimeras because of overexpression of oncogenes such as C-myc. Also, residual transgene expression alters
overall gene expression of the IPSCs, thereby making them different from human ES cells. 3 approaches to solve
these issues:

Transient expression of the reprogramming factors using adenoviral or transient transfection strategies;
replacement of the retroviral reprogramming factors by small molecules or prt. delivery; genomic targeting of the
reprogramming factors to a single locus using techniques such as LoxP recombinase. Reprogramming w/o
integrated reprogramming factors may be possible in the future.

Molecular Bases of Stemness

Blastomeres, Blastocyst, and the ICM: The zygote and the single blastomeres from a two or four-embryo
are totipotent. The outer cells of the blastocyst compact into the trophectoderm, from which the placenta will
derive. The inner cell mass (ICM) will give rise to the embryo proper (only), and its cells are thus considered
pluripotent. Once isolated and cultured in vitro the ICM may be propagated as an ES cell line.

Four Main Molecular Modules of Pluripotency: IL6 cytokine family, extrinsic determinants, intrinsic
determinants (i.e. TFs), and the epigenetic configuration of the cell

EC Cell Definition (again): First type pluripotent stem cells ever cultured. Self-renewing, undifferentiated
cells derived from teratocarcinomas. Teratocarcinomas form through the grafting of pre-implantation/pre-
gastrulation embryos or primordial germ cell lines (PGCs). Their one disadvantage is that since they are tumor
cells, they’re mostly aneuploid. Also, their contribution to the germ line has been an extremely rare event.

ES Cell Definition (again): Pluripotent cell derived from blastocyst stage mouse embryos. Their pluripotency
was demonstrated by their ability to form teratomas, and to diff. from embryoid bodies into tissues of all three
germ layers.

EG Cell Definition: Migratory PGCs form colonies that are morphologically indistinguishable from ES cell when
grown on feeder cells and a cocktail of growth factors incl. LIF (leukemia inhibitory factor), bFGF (basic fibroblast
GF), and SCF (stem cell factor). They have all the capabilities of normal ES cell, i.e. formation of the three germ
layers in vitro, teratoma formation in vivo, and contribution to the germ line if grafted into the blastocyst.

SC niche & ES cell niche comparison: Ordinary SC niche is hard to mimic, w/ lots of technical
complications; ES cells are easier to maintain in vitro, but a layer of fibroblast feeder cells is necessary for keeping
them pluripotent; this suggests that fibroblasts are necessary for maintaining self-renewal, suppressing diff., or
both.

LIF: Embryonic fibroblasts maintain the pluripotency of ES cells by secreting a factor known as leukemia inhibiting
factor, aka LIF (aka differentiation activity). LIF is member of the IL-6 family of cytokines, incl. IL6, oncostatin M,
ciliary neurotrophic factor (CNTF), and cardiotrophin 1 (CT1); they have a plethora of roles.
IL6 Family Receptor Structure & modus operandi: It belongs to the cytokine receptor class I family.
Extracellular domain: a variable number of fibronectin type III modules, 2 of which are conserved and constitute
the cytokine-binding site. Cytoplasmic domain: three conserved motifs: box 1, box 2, and box 3 (in a membrane
proximal to distal order); responsible for transmitting the extracellular signal into the cytoplasm, lack intrinsic
kinase activity.

Binding of the IL-6 family cytokine leads to homodimerization of gp130 or the heterodimerization of gp130 and
the cognate receptor. Both LIF and CT-1 bind to LIFR and induce LIFR/gp130 heterodimerization. CTNF engages
the LIFR/gp130 as a signaling complex through an association with CTNF/CTNFR; OSM engages the LIFR/gp130
or OSMR/gp130 by binding to the gp130 portion of the heterodimers. All the receptor-cytokine complexes share
gp130 module as a critical signal transduction module. The gp130 prt. is quite the cross talk point; LIFR, OSMR,
and CTNFR are all expressed in ES cells, consequently, CT-1, OSM, CNTF and LIF are interchangeable in supporting
ES cell derivation and maintenance of ES cells in culture. IL-6 & IL-11 cannot substitute, as their receptor is not
expressed in ES cells; however IL-6 can prevent ES cell diff. if injected along w/ a soluble from of the IL-6 receptor,
which is able to induce gp130 homodimerization. LIFR, CTNFR, and gp130 null mutations are lethal; they’re quite
critical.

Diapause: Lactating females can conceive while still nursing their pups, but cannot support blastocyst
implantation because they do not produce estrogen at (from) the fourth day of gestation. LIFR null and gp130
null embryos are unable to resume development after a few days of diapause. The number of gp130 null ICM cells
reduces gradually by gp130-absence-induced apoptosis. Delayed gp130 null blastocyst cannot form a pluripotent
outgrowth in vitro, as they differentiate exclusively into parietal endoderm.

Gp130 signal transduction:

Similar to the figure, the gp130 is the receptor in the shape (hetero- or homodimer). The docking sites created by
tyrosine-phosphorylation of gp130 are for SH2. The pathway then branches to several pathways involving STAT1
& STAT2 (gene expression regulation), ERK1/ERK2 (extracellular signaling receptor complexes) or MAPK (i.e. the
Ras pathway), and PI3K.

STAT 3: Plays a crucial role that cannot be compensated for by other members of the stat family. The activation
of STAT 3 is enough to maintain the undiff. Phenotype of ES cells. Yet, the LIF-STAT3 pathway is not the only
pathway capable of this feat. Another factor, called ES cell renewal factor, was shown to operate quite
independently of the former pathway to maintain ES cell undiff. phenotype.

LIF-MAP kinase pathway: The bridging factor between LIP and MAP is the tyrosine phosphatase SHP2.
Actually inhibits self-renewal signaling pathways and promotes diff. Activated ERKs undergo nuclear
transportation, which enables them to regulate gene expression for proliferation, diff., and cell survival. SHP2
interacts w/ ERKs by the scaffold prt.s Grb2 and Gab1. SHP2/GRB2 association leads to Ras pathway through the
monomeric GTPase SOS. Another route is via Gab1 and PI3K to Ras.

Grb2 connects SHP2 and SOS. Grb2 itself is activated by gp130 the Grb2-Ras-MAPK pathway is essential for early
specification of the endoderm tissues.

Summary of the pathways so

far:

FGF4 and Erk: It’s been demonstrated that the major activator of the Erk pathway for ES cell maintenance in
mice is fibroblast growth factor 4
The Canonical Wnt Pathway: results in sustained
expression of the pluripotent markers Oct4 and Nanog.

The Convergence of the Wnt and LIF pathways: They

respectively activate gene transcription and stabilize cMyc.

BMP4: LIF is not enough for maintaining ES cells in serum-free culture. The identified necessary factor in serum
was BMP4; which maintains self-renewal and inhibits neural diff of ES cells. BMP acts via the smad pathway:
The PI3K-Akt pathway:
Also induced by LIF. Also has
crosstalk w/ the Wnt pathway
through the Akt mediated
phosphorylation (inhibition)
of GSK3β.

Integrative ES Cell Maintenance Procedure:

Culture either containing serum or a combination of LIF and
BMP.

Oct4: Essential in the establishment of ICM pluripotency. In

its absence, the interior cells of the pre-implantation embryo
are diverted to a trophectoderm fate. Its amount has been
proven to be important in determining cell fate:
Notch Pathway: