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lezione BCAB del 04-10-2013, 10-10-13 e 11-10-13

Isabelle Perroteau

on 6 October 2014

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Transcript of stem_cells

Where from?
What’s for?
Where are we?
It is now well accepted that a stem cell must fulfill three criteria:

1- it must be capable of self-renewal, i.e., undergoing symmetric or asymmetric divisions through which the stem cell population is maintained.

2- A single cell must be capable of multilineage differentiation.

3- In vivo functional reconstitution of a given tissue.

Where from?
What's for?
Clinical trials
Where are we?
Stem Cells
The definition of ‘stem cell’ is essentially
: ‘‘rather than referring to a discrete cellular entity, a stem cell most accurately refers to a biological function that can be induced in many distinct types of cells, even differentiated cells’’

various types of cells associated with stemness
- Embryonic stem cells
- Adult stem cells
- Artificially reprogrammed stem cell
- Cancer stem cells

Do they have something fundamental in common?
Are we applying a common name to very different entities?

Stem cells (SCs) are
very small
cells that do
t have the
phenotypic characteristics
of cells from any known adult tissue (epithelial, connective, muscle, neural, and immune) but are
able to generate ‘de novo’ differentiated cells
of the types found in any of these tissues.

There are five defined types of SCs:
physiological SCs that are present at different stages of life
Embryonic stem cells (
Adult stem cells (
engineered or ‘induced’ SCs
pathological cells present in cancers and having some stem propertie
Cancer SCs (
Induced pluripotent stem (
) cells)
Many investigations have been carried out to define the
essential characteristics of stemness
Such properties should be common to all known SCs, or at least to cells making up one of the four groups described earlier, and refer to the following functional aspects:

1) expression of
of pluripotency,
2) activation of
signal transduction pathways
that maintain stemness,
3) a characteristic
proliferative state
4) how the pluripotent SCs
in vitro
5) how they behave after reintroduction to an
in vivo
Embryonic stem cells (ESCs)
ESCs make up the
inner cell mass (ICM)
of the blastocyst before implantation or before any commitment to embryonic cell fates is detectable at the molecular level. They are the most studied SCs and knowledge obtained from ESCs has guided the investigations of other types of SC.
In human
nuclear transfer ESCs (
A nerve cell (the right) derived from mouse undifferentiated cells (the top left) via an embryoid body (an assembly of the undifferentiated cells, the bottom left) in vitro.
Hanging drop culture. Embryoid bodies for the differentiation of embryonic stem cells in the embryonic stem cell test are generated by pipetting a single-cell suspension onto the lid of a cell culture dish. The cells aggregate at the bottom of the drop by gravitational force, thereby forming the embryoid body.
2nd step:
Embryoid bodies
3rd step: terminal differentiation
Alternative to hanging drop:
Low cell binding plates/dishes HydroCell
Key features of HydroCell®

♦Super hydrophilic polymer is fixed to the surface of the dish/plate at nano-thickness level.
♦The surface is highly cells/protein resistent
Improved macrophage culture "in vitro".
Embryoid body formation from ES cells.
Spheroid formation in culture.
Engineered or ‘induced’ SCs
Embryonic stem cells obtained by nuclear transfer
Embryonic stem cells obtained by nuclear transfer are genetically identical to the patient ---> no immune
Adult stem cells
Adult stem cells from the CNS
The most extensively studied adult stem cell is the
hematopoietic stem cell (HSC)
Neural stem cells (NSC)
give rise to neurons, astrocytes, and oligodendrocytes.
Mesenchymal stem cells (MSC)
differentiate into fibroblasts, osteoblasts, chondroblasts, adipocytes, and skeletal muscle. Other stem cells have been identified, including
gastrointestinal stem cells, epidermal stem cells, and hepatic stem cells
(also called oval cells).

Hemopoietic stem cell
Bone marrow transplantation
Heart stem cell
Mesenchymal stem cell (MSC)
mesenchymal stem cells (MSCs) in the bone-marrow cavity to self-renew (curved arrow) and to differentiate (straight, solid arrows) towards the mesodermal lineage. The reported ability to transdifferentiate into cells of other lineages (ectoderm and endoderm) is shown by dashed arrows, as transdifferentiation is controversial in vivo.
The stem cell niche
The adult SC niche. ASCs are organized in a compact structure supported by MSCs and receiving specific nervous (sympathetic) and vascular support.
ASCs maintain a basal slow proliferative rate
that neutrally drives the cells either to remain in the niche or become converted into progenitors and leave the niche. Randomly occurring factors such as proximity to cytokines might decide ASC fate through symmetric or asymmetric divisions.
(A) With
division asymmetry
, stem cells (gray) always divide to produce one stem cell daughter (gray) and one differentiating daughter cell (purple). The same number of stem cells is present both before and after division.

(B) With
population asymmetry
, stem cells undergo either symmetric renewal to produce two stem daughters (gray) or symmetric differentiation to produce two differentiating daughters (purple) at equal frequencies. Overall, as the stem cell population divides, the number of stem cells remains constant.
Maintenance of stem cell populations
by division asymmetry or population asymmetry.
Stem cell behaviour, in particular the balance between self-renewal and differentiation, is ultimately controlled by the
integration of intrinsic factors with extrinsic cues
supplied by the surrounding microenvironment, known as the stem cell niche. The identification and characterization of niches within tissues has revealed an intriguing conservation of many components, although the mechanisms that regulate how niches are established, maintained and modified to support specific tissue stem cell functions are just beginning to be uncovered
Neutral competition in stem cell niches: number of stem cells is maintained
(A) Under neutral competition, all stem cells are equally likely to be lost from the niche. Symmetric differentiation results in loss of a stem cell, whereas symmetric renewal results in gain of a stem cell. The rates of symmetric differentiation and symmetric renewal must be balanced to ensure the maintenance of a stable stem cell population.
(D) The small intestinal crypt contains Paneth cells (green) at the base, interspersed with Lgr5+ intestinal stem cells (ISCs) (gray). ISCs produce transit-amplifying progeny (purple) that migrate up the crypt and then up the villi to regenerate the intestinal epithelial layer. A cross-section of the crypt just above the stem-cell compartment (dashed line) shows a ring of transit-amplifying cells (right image). These are progeny from multiple ISCs. If an ISC is genetically marked (blue), this mark will subsequently appear in the transit-amplifying stem cell progeny (blue, in cross-section).
Multicolor labeling of the ISCs in each crypt confers permanent
fluorescent labels of four different colors
, at random, to the ISC population. Initially, cells in the transit-amplifying cross-section are heterogeneously labeled . As ISCs either symmetrically differentiate (straight arrow) and leave the niche or symmetrically renew (curved arrow) to produce replacement stem cells, labeling in the cross-section becomes more homogeneous. Eventually, the niche will become monoclonal with regard to a label (right).
This path to homogeneity is random with regard to which label becomes dominant
. Marked clones exhibit scaling behavior until the crypt reaches monoclonality.
Induced pluripotent stem cell from adult cell
o transdifferentiation
Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells).
A siRNA screen for factors that can boost epigenetic reversion of primed EpiSCs into naive ES cells identified Mbd3 as a potential candidate.
Single-cell reprogramming efficiency and quantification for EpiSC reprogramming from different mutant lines. The pBRY-Mbd3 rescue construct was stably expressed in the indicated lines (n = 4)
Mbd3 is a key component in the NuRD complex, ubiquitously expressed in all somatic cells which can mediate gene repression through histone deacetylation and chromatin remodelling activities
the reprogramming factors Oct4, Sox2 and Klf4 recruit not only transcriptional coactivators (such as Tet2)5 to stem-cell genes, but also transcriptional repressors, including Mbd3, which potently inhibit gene reactivation
Constructs encoding Flag-tagged OCT4, SOX2, KLF4, MYC, Nanog or HDAC1 were transfected into HEK293T cells in combination with Mbd3. The cell lysates were immunoprecipitated (IP) with an anti-Flag antibody (or anti-IgG as control), followed by an immunoblot analysis (IB) (n = 3 biological replicates).
Eliminating Mbd3 enables the coactivators to efficiently resuscitate dormant stem-cell genes, allowing almost all cells to be converted into stem cells.
Stem-cell niches in mammalian tissues are often heterogeneous and compartmentalized; however, whether distinct niche locations determine different stem-cell fates remains unclear.
Cancer stem cells

"Stem-cell populations are established in 'niches' —
specific anatomic locations
that regulate how they participate in tissue
maintenance and repair
The niche saves stem cells from depletion, while protecting the host from over-exuberant stem-cell proliferation. It constitutes a basic unit of tissue physiology, integrating signals that mediate the balanced response of stem cells to the needs of organisms. Yet the niche may also induce pathologies by imposing aberrant function on stem cells or other targets. The
between stem cells and their niche creates the
dynamic system
necessary for sustaining tissues, and for the ultimate design of stem-cell therapeutics...The simple location of stem cells is not sufficient to define a niche. The niche must have
both anatomic and functional dimensions
—David T. Scadden, The stem-cell niche as an entity of action, Nature, 441 (7097), 1075-1079 (29 June 2006)
The adult stem cell "niche"
Definition of "niche"
Adult Stem Cells
Mesenchymal SC
Parenchymal SC
New somatic non connective tissues
New somatic connective tissues
Extensively distributed
Agregate in the organs in structures called "the niche"
The niche does not exist during embryonic development, it is specific of ASC
Until now, only a few SC niches have been identified, including the bottom of the
intestinal crypts
, the limbal zone of the
, the
subventricular zone
in the brain and the
hair follicle bulge
Human derived SC on animal models
In models of stroke, similar functional recovery has been achieved after injection of MSCs into the carotid artery or intravenously, although substantially fewer cells incorporated into damaged brain issue via the i.v. route
Many of the early studies using autologous SC injections in patients did not have adequate controls or were not double-blinded. The current scenario is very different, with about 3400 controlled clinical trials registered as ‘Cell Therapy’ in the NIH database. Of these, nearly 1900 have now finished, although only some 125 have presented their results. Many of these clinical studies are phase I trials relating to safety issues and many use autologous bone marrow purified populations. A few hundred are using MSCs, although none has yet formally presented their results; some trials are using skin derivatives grown in vitro.
Currently, among 50 trials are endocrinology related, of which all are using cell therapy in diabetic patients but none have yet presented their results.
Clinical trials using cell therapy
Concerns about safety in all these trials have increased since early reports of donor-derived leukemia after bone marrow transplants and one case of multiple neural tumors in the spinal cauda equina and brainstem meninges arising 5 years after repeated transplantation of fetal neural precursors (Greaves 2006, Amariglio et al. 2009). It has therefore been proposed that cells used in therapy should have an inherent trigger for destruction if necessary, and a modified inactive caspase 9 transgene has recently been introduced into human T-cells. This protein is fused to an FK protein domain and needs access to a smallmolecule drug to dimerize and become active, inducing apoptosis. The modified T-cells have been injected into five leukemia patients who previously received a matched isotypic bone marrow transplant and have been detected and found to be functionally active in peripheral blood. Indeed, when four of these patients developed graft-vs-host disease, as expected in many transplant patients, a single injection of the dimerizing drug was enough to control the disease and prevent its recurrence (Di Stasi et al. 2011).
A recent study used an oncolytic virus, an adenovirus bearing mutated oncogenes that specifically arrests and kills cancer cells, to transduce autologous MSCs, which were then injected into four children with grade IV neuroblastoma resistant to therapy. MSCs were recruited and migrated into the tumor. In one of the young patients, the virus destroyed enough tumor cells to generate a CD8 cytotoxic response, the tumor disappeared and the patient was free of the disease (Garcia-Castro et al. 2010). Improving oncolytic viruses and adjusting the doses of injected MSCs could therefore have potential as be an alternative therapy in some advanced cancers in the future.
partial locomotor recovery after spinal damage
Hit-and-Run Action of Stem Cells Exploited for Targeted Drug Delivery
Mesenchymal stem cells (MSCs) possess a set of several fairly unique properties which make them ideally suited both for cellular therapies/regenerative medicine, and as vehicles for gene and drug delivery. These include: 1) relative ease of isolation; 2) the ability to differentiate into a wide variety of seemingly functional cell types of both mesenchymal and non-mesenchymal origin; 3) the ability to be extensively expanded in culture without a loss of differentiative capacity; 4) they are not only hypoimmunogenic, but they
produce immunosuppression upon transplantation
; 5) their pronounced
anti-inflammatory properties
; and 6) their ability to
home to damaged tissues, tumors, and metastases
following in vivo administration.
Mesenchymal stem cells can be used in regenerative medicine, but also as immunomodulatory/anti-inflammatory agents, and as vehicles for transferring both therapeutic genes in genetic disease and genes designed to destroy malignant cells.
Components of stem cell niches:
the stem cell itself,
stromal cells,
soluble factors,
extracellular matrix,
neural inputs,
vascular network
cell adhesion components

note that although many niche components are conserved, it is unlikely that every niche necessarily includes all of the components listed. Instead, niches are likely to incorporate a selection of these possible avenues for communication, specifically adapted to the particular functions of that niche, which might be to provide structural support, trophic support, topographical information and/or physiological cues
Targeting stem cell niche for therapy
in light of accumulating evidence suggesting that tumour-propagating cancer stem cells are dependent on signals from their niche, just like their non-malignant counterparts, therapeutic ablation of components of the cancer stem cell niche could provide a novel strategy to remove tumour support factors, and thus achieve cancer remission

Le cellule di partenza sono senza dubbio differenziate in quanto sono linfociti (unico tipo cellulare in cui il differenziamento avviene per ricombinazione genica)
STAP rispetto a iPS:

Più rapido
Più efficacia (resa più elevata)
Potenzialità maggiori
Meno rischioso

Le cellule staminali neonatali ottenute per stress cellulare (pH acido) sono state rese fluorescenti e iniettate in un embrione di topo: partecipano alla produzione di tutti tipi cellulari compresi gli annessi extraembrionali (placenta)
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