Self-renewing (or highly regenerative) tissues and the function of adult stem cells

Many adult tissues such as the skin epidermis, the gastrointestinal epithelia or the hematopoietic system are regenerative. In such tissues mature cells such as the stratum corneum of the skin, differentiated enterocytes at the tip of the intestinal villi or blood erythrocytes have rather short half-lives and must be continuously produced to replenish the large number of cells steadily lost by shedding or apoptosis. Life-long production of differentiated progeny relies on the activity of rare long lived adult tissue stem cells, which have the ability to both perpetuate themselves through self-renewal and to generate all mature cell types of a particular tissue through differentiation (Fig. 1). Stem cells are thought to be located in a special microenvironment called the stem cell niche, which prevents differentiation and maintains stem cell activity by poorly understood mechanisms (Fig. 2 and Fig. 4 top) (Wilson and Trumpp, 2006). Upon division two daughter cells are generated, one of which, on average, stays in the niche maintaining its stem cell identity. In contrast, the other daughter cell eventually leaves the niche consequently losing the capacity to self-renew and acquiring the fate of a transit amplifying cell (TA‑cell) (Fig. 1). The function of these cells is to rapidly expand through proliferation while simultaneously differentiating along one of the lineages present in the given tissue. Continuous differentiation correlates with progressive loss of multipotency. This ends by exit from the cell cycle allowing maturation into terminally differentiated cell types such as enterocytes, erythrocytes or terminally differentiated skin keratinocytes (Fig. 1). In case of injury quiescent stem cells are activated and recruited to the site of injury and ensure rapid tissue repair for example after blood loss or skin damage. In striking contrast, non-self renewing tissues such as brain, lung, heart or kidney respond to injury with extensive scaring, remodeling and often loss of tissue function.

Figure 1: The maintenance of self-renewing adult tissues (i.e. intestinal mucosa, skin epidermis or the hematopoietic system) requires the life‑long production of differentiated progeny, which relies on the activity of rare long lived adult tissue stem cells. These stem cells have the capacity to self-renew and give rise to transient amplifying (progenitor) cell types leading to massive expansion of organ specific cell. Upon terminal differentiation functional mature cells are produced ensuring the maintenance of organ specific physiology.

Figure 2: Bone marrow HSC niche. Right: Hematopoietic stem cells (HSCs) are mainly located in the trabecular part of the long bones. The endosteum lines the inner bone surfaces and is comprised of stromal cells and osteoclasts (white) as well as spindle shaped osteoblasts (brown). The latter are thought to serve as niche cells to maintain quiescence and prevent differentiation of attached HSCs. In response to injury HSCs can be mobilized resulting in the migration of HSCs to peripheral hematopoietic organs such as the spleen and the liver. HSCs in the circulation can home back to the endosteal niche a process called lodging. Left: Purified and fluorescently (CFSE) labeled stem/progenitor cells can be detected at the endosteum 15hrs after transplantation (Wilson et al., 2004). An HSC (green) is in direct contact with an osteopontin secreting (OPN-red, nuclei-blue) osteoblast at the endosteum comprising a stem cell niche (see also Wilson and Trumpp, Nature Reviews Immunology 2006)

The cancer genes c‑myc and pten play crucial roles in adult stem cells

Adult stem cells likely represent a major target for tumor initiation and gene therapy. Therefore, understanding the regulation of normal stem cell self-renewal is also fundamental to understand the regulation of cancer cell proliferation, because cancer can be considered to be a disease of unregulated self-renewal. Our research focuses on the elucidation of the molecular basis of stem cell self-renewal and differentiation as well as the identification of cancer stem cells in vivo. For this we take advantage of a number of mouse models which are mutant for two of the most frequently affected genes in human cancers, c‑myc  (Fig. 3) and Pten.

Figure 3: c‑myc belongs to the class of immediate early genes. Its expression is induced in response to a number of mitogenic signals. One out of five human cancers carry mutations of the c‑myc gene, which encodes a short lived nuclear protein c‑Myc that can activate or repress transcription of a specific set of still elusive target genes. Genes activated by c‑Myc are antagonized by Mad/Max. Expression of the transcription factor c‑Myc leads to the activation or repression of target genes which affect a number of biological processes.

Both cancer genes seem to play non-overlapping roles in stem cell function. We have recently shown that loss of Pten in the brain leads to an increase in neuronal stem cell number due to increased survival, proliferation and growth (Groszer et al., 2001). In contrast loss of c‑Myc in the bone marrow (BM) does not affect proliferation of hematopoietic stem cells (HSC), but blocks stem cell differentiation and appears to have a direct role in controlling the balance between self-renewal and differentiation. This leads to an increase of the HSC pool while differentiating cell types are lost providing the mechanism for the severe anemia observed in those mutants (Wilson et al., 2004 and Wilson and Trumpp Nat. Rev. Immunol. 2006). The increased number of stem cells is apparently due to a change in the interaction between the mutant HSCs and their niche (Fig. 4).

Figure 4: Model of how c‑Myc expression controls the balance between hematopoietic stem cell (HSC) self-renewal and differentiation by regulating retention or departure of stem cells from the differentiation preventive niche environment (brown shading). Top: A quiescent HSC expressing low c‑Myc levels is anchored to stem cell niche cells by homotypic interactions (red block) and by interactions with the niche extracellular matrix (blue block). Signals from the niche (red arrow) maintain the « stemness » of the HSC by promoting self renewal and preventing differentiation whereas signals from the HSC (blue arrow) are required to maintain the function of the niche. In response to mitogenic signals the HSC enters the cell cycle and generates two daughter cells. (a) In the absence of c‑Myc induction integrins, cadherins and other putative cell adhesion molecules remain highly expressed retaining both daughter cells in the niche, thereby promoting expansion of HSCs at the expense of differentiation. (b) Induction of c‑Myc in only one of the daughter cells generates asymmetry with one HSC retained in the niche and one leaving the niche promoting differentiation into committed progenitors (CP). In this homeostatic situation the stem cell pool is maintained while differentiated progeny is produced. (c) High Myc expression in both daughter cells (e.g. ectopic c‑Myc expression in HSCs) results in repression of cell adhesion and departure of cells from the niche. This leads to the production of two CPs and hence progressive exhaustion of the stem cell pool due to differentiation.

Identification and characterization of “cancer stem cells” in primary tumors

Despite major advances in basic cancer research, and especially in understanding the molecular and biochemical pathways that are involved in tumorigenesis and malignant transformation, there hasn’t really been much significant progress in cancer therapy since years. Could this suggest that conventional strategies for confronting cancer have been exhausted? A tumor can be viewed as an aberrant organ initiated by a tumorigenic cancer cell that has acquired the capacity for indefinite proliferation through accumulated mutations. In searching for the cancer-originating cell type, apparent analogies between tumorigenic cells and normal stem cells have long been recognized even before the discovery of oncogenes and tumor suppressor genes twenty-five years ago (reviewed in Reya et al., 2001 and Dean et al., 2005). The aim of this program, which we have recently initiated, is to obtain evidence whether solid tumors are driven by a small number of genetically altered stem cells called “Cancer Stem Cells” (CSC) (Fig. 5). We have recently started to develop techniques for the identification, isolation and characterization of CSC from primary murine and human cancer. Using technologies related to stem cell enrichment methods using sophisticated cell sorting (FACS) technologies (e.g. “side population”) in combination with specialized stem cell maintaining culture conditions allowed us to enrich tumor initiating cells, putative CSC. As the most stringent functional test for the tumor initiating potential we use orthotopic grafts of enriched CSC cells into NOD/SCID or RAG2^‑/‑; gC^‑/‑ mice. Comparison of the transcriptome of purified CSC and tumor cells that have no tumor initiating activity will in the future allow the isolation of specific pathways engaged in CSC. This may enable us to specifically target and destroy CSCs, thus preventing tumor relapse after chemotherapy (Fig. 5).

Figure 5: Conventional therapies may shrink tumor mass, but spare cancer stem cells (CSC). CSC are resistant and remain viable and after some time re-establish the tumor. By contrast if therapies can be targeted against CSC, then they might render tumors unable to maintain themselves to grow. Thus, therapies that target CSC should not shrink the tumor immediately but may eventually lead to tumor degeneration. Combination of conventional therapies with drugs that specifically target CSC should lead to fast and long lasting cures.

Future studies will further extend the use of mouse models to study stem cell self-renewal in various adult tissues such as skin and intestine as well as in the hematopoietic system. Furthermore we have begun to visualize self-renewal divisions of highly purified normal and mutant HSCs by time lapse video microscopy and are using normal and mutant stem cells for genome wide Affymetrix microarray analysis to identify novel genes involved in stem cell self-renewal. Our aim is to use these insights to develop strategies that would allow expansion of adult stem cells in vitro and obtain insights into the pathological events that lead to the generation of cancer stem cells.

Our goal is to use mouse molecular genetics to understand:

• the molecular mechanism that controls the balance between stem cell self-renewal and differentiation
• the molecular and cellular nature of a “stem cell niche”
• the role of c‑Myc and Pten in stem cells, differentiating cells, organs and tumors
•  the molecular and cellular difference between “cancer stem cells” and normal stem cells.

Collaborations

Our group collaborates with: Didier Trono (EPFL); Friedrich Beermann (ISREC); Jörg Huelsken (ISREC), Anne Wilson and Rob MacDonald (Ludwig Institute for Cancer Research, Lausanne), Jürg Tschopp, Ivan Stamenkovic (University of Lausanne), Ivan Martin (Basel), Bruno Amati (Milano), Stefano Piccolo (Padua), Pierre Chambon (Strasbourg), Dirk Eick (München), Martin Eilers (Marburg), Sylvie Robine (Paris), Elisabeth Robertson (Cambridge), Yosef Refaeli (Denver), Hong Wu (Los Angeles), Steven Martin (Berkeley), Scott Kogan (San Francisco), Boris Bastian (San Francisco).

Keywords

Organism:    
Mouse (mus musculus), human tumor samples
Systems:      
Hematopoietic system, leukemia, lymphoma, skin epidermis, intestinal    mucosa, liver, ex vivo 2D and 3D culture systems
Methods:      
Stem cell assays (in vivo and in vitro), mouse genetics, gene targeting, Cre/loxP system, FACS, tumor assays, bone marrow reconstitution, retroviruses, lentiviruses, Affymetrix microarrays, ex vivo cell culture, time-lapse video microscopy, histology, RNA and protein expression studies in vitro and in vivo
Molecules:   
c‑Myc, N‑Myc, Pten, p21CIP1, Wnt, hedgehogs, cadherins, type I interferons