In the first part of this work, we showed that human MSC derived from adult and pediatric bone marrow express similar surface markers and can differentiate into mesodermal tissue. Expansion potential was slightly higher for pediatric than for adult MSC, but none expressed stem cell markers such as oct-4 (data not shown) nor did we detect telomerase activity in these cells. This is in accordance with previous studies (Baxter, 2004; Bernardo, 2007b; Zimmermann, 2003). Nevertheless, in the in vitro differentiation experiments, albumin and alpha1 anti-trypsin (API) were more frequently expressed in pediatric than in adult MSC after co-culture with Huh7 cells.
We could not demonstrate albumin expression at the protein level, after co-culture. In pediatric MSC, expression of albumin and API were induced even without growth factor supplementation in the culture medium. The higher frequency of albumin expression in pediatric MSC (5/8 vs 2/10 independent experiments) and differentiation in the absence of growth factors suggested that pediatric MSC have a stronger hepatogenic differentiation potential.
Age interferes with multiple biological processes: telomere length (Guillot, 2007), accumulation of possible DNA damage, and accumulation of ROS, thereby increasing risk of degeneration (Bernardo, 2007b; Li, 2007b; Rubio, 2005). In vitro expansion, to obtain a high amount of MSC for possible clinical application, also harbors the risks of “artificial aging” with decreased differentiation potential and increased risk of degeneration towards cancer (Rubio, 2005; Rubio, 2008).
When cultured in Huh7-conditioned media, without growth factors, MSC expressed alpha-smooth muscle actin, and vimentin, but did not express albumin. In the absence of HuH7 cells, Huh7-conditioned medium alone induced a fibrogenic potential, with expression of a marker of myofibroblasts (Gabbiani, 1983). The identification of factors secreted by Huh7, possibly in response to molecules secreted by MSC and responsible for the expression of albumin in co-culture experiments will be important to better understand the differentiation processes. On the other hand, identification of fibrogenic factors in Huh7-conditioned media would also be of major importance to prevent scaring and differentiation into myofibroblasts. Identification of these molecules may also allow to potentially inhibit these pathways and reverse fibrosis.
It was reported that hepatocytes injected into the spleen spontaneously migrate to the liver in the portal flow after partial hepatectomy (Nguyen, 2006; Nguyen, 2002).
However, in our conditions, after intrasplenic injection, very few MSC migrated to the liver and most engrafted within the splenic parenchyma. This may be explained by an inopportune timing or inadequate liver injury, with signals too weak for MSC to respond and home into the liver. Another reason may be transplantation of inappropriate cells, with reduced expression of chemokine receptors or inadequate response to cytokine signaling from the liver, as in other studies MSC from the bone marrow were able to migrate in a CXCR4-dependent mechanism (Son, 2006; Sordi, 2005).
In order to circumvent the problem of migration and to further expose MSC directly to liver parenchyma, we injected MSC directly into the remnant of liver parenchyma, after partial hepatectomy. Cells engrafted within the parenchyma, and retained spindle shaped morphology. Immunofluorescence against vimentin and Masson’s trichrome staining showed that engrafted cells co-localized with collagen deposits.
After analyzing all transplanted mice liver tissue, by immunohistochemistry and by RT-PCR, no human albumin could be detected. We concluded that adult and pediatric human MSC do not differentiate into hepatocytes in vivo, confirming results obtained in rats by Popp et al (Popp, 2007), but in opposition to other published studies (Aurich, 2007; Banas, 2007; Campard, 2008; Lysy, 2008; Lysy, 2007; Najimi, 2007; Oyagi, 2006; Seo, 2005). Most models used expansion of MSC before transplantation, thereby possibly limiting differentiation potential (Banfi, 2002).
Inducing fibrogenic tissue or contributing to endogenous fibrosis may be harmful for clinical outcome. The absence of hepatic differentiation after intraparenchymal injection may be related to: 1) insufficient / inappropriate injury of the liver, with inappropriate timing of injection, with inadequate growth factor secretion, or secretion of fibrogenic growth factors such as TGFbeta, instead of hepatogenic growth factors, 2) transplantation of cells with limited differentiation potential, that can be related to culture conditions, expansion-induced damage, age of donors, senescence, 3) discrepancies between growth factors secreted within a mouse liver and human cells, related to possible differences of expression regulation and protein structures between species.
Furthermore, in vivo studies have demonstrated that frequency of engraftment and differentiation into hepatocytes remains low and insufficient to restore normal function in acute hepatic failure, and also too slow to serve as a bridge to orthotopic liver
transplantation or to sustain function until endogenous regeneration (Aurich, 2007;
Banas, 2007; Campard, 2008; Oyagi, 2006; Seo, 2005). Second, transplantation of a potentially fibrogenic cell type may worsen the disease, especially in a liver which is already undergoing chronic damage, scarring and partial regeneration, as it is the case during cirrhosis (di Bonzo, 2008).
Pre-differentiation of MSC prior to transplantation, by culture in differentiation medium has been described and showed better engraftment and in vivo function than undifferentiated cells (Banas, 2007). We did not pre-treat our cells for several reasons. First, we wanted to investigate the hepatic differentiation potential in vivo.
Second, production of several million of differentiated cells for animal models necessitates large quantities of growth factors, which are costly. Third, if this technology is applied to humans, generation of several hundred million cells would require large bioreactors.
3.2. Future perspectives of MSC in hepatic replacement
In continuation of our experiments, further research is required to identify possible fibrogenic factors in vitro and in vivo. First, Huh7-conditioned medium should be analyzed for its composition in cytokines and chemokines and compared to control medium. A proteomic approach may identify differences within these media. Factors regulating expression of alpha-smooth muscle actin should also be investigated, and possibly identified within Huh7-conditioned medium. Identification of molecules improving migration and differentiation into hepatocytes in vitro should also be further studied, possibly using drug/cytokine/growth factor screening methods, already applied to ESC (Suter, 2009).
In vivo, understanding the importance and nature of interactions of MSC with hepatocytes and endothelial cells within the liver would allow identifying mechanisms implicated in engraftment and possible differentiation. Present technology using in vivo cell-tracking, either by transduction of luciferase or by labeling MSC with iron particles may help to understand homing and migration characteristics. Further basic research on the fibrogenic potential of MSC is required. The induction pathways of alpha smooth muscle actin expression might be different in fibroblasts and MSC.
Identification of molecules contributing to this process in vitro, as well as within pathological liver parenchyma are required with the aim to control its expression and possibly prevent differentiation into myofibroblast and thereby fibrosis.
3.3. Second publication: discussion
We identified fibroblast-like cells within exocrine tissue of the pancreas, with a phenotype and differentiation potential similar to MSC isolated from the bone marrow. These cells could be expanded up to 40 population doublings. These cells expressed markers of beta-cell progenitors, such as Pdx1, Nestin, Nkx2.2, Nkx6.1, NeuroD, Isl1, and Ngn3. Insulin expression was first present during expansion, due to contamination by beta cells, present within exocrine enriched tissue. Insulin disappeared after 3 passages, whereas expression of beta cells transcription factors was maintained.
Vimentin and nestin were expressed by a majority of pancreatic MSC, demonstrating their mesenchymal phenotype and possible beta cell progenitor characteristics (Eberhardt, 2006; Zhang, 2005b; Zulewski, 2001). Nestin expression and its role in pancreas progenitor remains controversial, and its use as stem cell markers has been questioned recently (Street, 2004; Vaittinen, 2001). However the expression of multiple beta cell transcription factors strengthens the hypothesis that endocrine progenitors similar to MSC can be derived from pancreatic endocrine tissue. These cells did not express CA-19-9, a marker for ductal cells, but retained CK19 expression, thus suggesting an epithelial origin.
MSC were described in multiple tissues, of mesodermal and non-mesodermal origin.
Our findings also support the hypothesis that MSC are distributed throughout the body and reside in a ubiquitous niche (da Silva Meirelles, 2008; da Silva Meirelles, 2006).
To induce differentiation into endocrine cells able to express insulin, pancreatic MSC were cultured at high density on non-adherent plastic in CMRL medium containing nicotinamide, Activin A and followed by the addition of HGF. Cell clusters, resembling islet structures, spontaneously formed and expressed insulin and glucokinase, whereas cells in control medium did not express markers of differentiated beta cells.
Glut2 expression was not induced and insulin was not detected at a protein level, suggesting a partial differentiation.
Gershengorn’s group showed recently that in progenitors derived from pancreas tissue, histone modification in regard of the insulin gene is similar to modifications found in islets. These chromatin modifications suggest that the insulin gene is in an active state in progenitors isolated from the pancreas, and may thereby facilitate insulin gene expression (Mutskov, 2007).
Seeberger et al isolated a similar type of MSC from ductal/exocrine pancreas tissue and demonstrated albumin expression after culture in hepatogenic medium and insulin expression after culture in endocrine differentiation medium (Seeberger, 2006). They did not observe insulin or albumin expression at protein level. In their differentiation experiments, Pdx1 and Ngn3 were not always expressed, while insulin was present. Both studies used tissue enriched in exocrine cells and depleted of islet. It was however not demonstrated whether MSC were derived from contaminating islets, vascular cells found within the pancreas, or from exocrine structures. MSC isolated from human pancreas reported by several groups may be cells with similar characteristics (Baertschiger, 2008; Davani, 2007; Seeberger, 2006). Further investigations are required to evaluate differentiation potential of MSC and especially their capacity to mature into beta cells suitable for clinical applications.
MSC can also interact with islets, in an undifferentiated state, as possible support.
Co-culture of bone marrow-derived MSC with islets and endothelial cells improved islet survival in vitro and increase revascularization (Johansson, 2008). Bone marrow-derived MSC are therefore of great interest for transplantation as they associate this trophic effect with immunomodulatory characteristics. Future research will show whether these characteristics are shared by pancreatic MSC.
3.4. Future perspectives of pancreatic MSC
Most studies using cells from poly-clonal cultures, one major aim for future studies would be to identify the exact phenotype of pancreatic progenitors and derive them clonally. The precise origin of pancreatic MSC remains controversial. Together with others, we hypothesize that they are derived from exocrine tissue (Baertschiger, 2008; Seeberger, 2006; Yatoh, 2007). Gershengorn’s and other groups claim that they are found within islets (Davani, 2007; Eberhardt, 2006; Morton, 2007). Some studies state that MSC can be found in all post-natal organs in the perivascular niche (da Silva Meirelles, 2008; da Silva Meirelles, 2006), whereas others suggest that MSC migrate from the bone marrow into peripheral organs after injury (Lee, 2006;
Sordi, 2005).
During islet isolation and purification, exocrine tissue always contaminates islets and vice-versa. The definitive demonstration of the origin of MSC within the pancreas will only be given once single cell culture with previously identified lineage markers can be traced in vivo.
Another future direction of research would consist in improving our knowledge of epigenetic modifications of these stem cells and possibly intervene at that level to facilitate expression of beta cell proteins. Mutskov et al already showed differences between bone marrow-derived MSC and pancreatic progenitors (Mutskov, 2007) in histone methylation of chromatin in regard of the insulin gene. Whether intervention on multiple beta cell genes to induce modification of chromatin into an open, active state is possible, through addition of growth factors to the medium or lentiviral-vector mediated transduction, remains to be demonstrated. Since 2006, knowing the capacity of fibroblasts to be reprogrammed into ESC-like cells, named IPS cells, questions dramatically the use of adult stem cells (Nakagawa, 2008; Takahashi, 2007a; Takahashi, 2007b). Differentiation of IPS into islets has not yet been demonstrated and, risks of teratoma associated with pluripotency remains a concern.
Improvement of differentiation protocols is an important point for future research to increase the number of cells that differentiate within a given population. Expression profiles and information on regulation of various genes during development and differentiation of ESC and adult stem cells will probably help to improve these protocols. Current research on multiple drugs and molecules in screening experiments performed on ESC and MSC may identify new agonists and antagonists of major signaling pathways to improve differentiation.
Finally, no information about immunomodulatory potentials of pancreas-derived MSC is available. Whether these cells express IDO, iNOS or other enzymes able to modify immune response can be hypothesized, as they share major characteristics with MSC. If they downregulated T-cells responses, their use for clinical transplantation as beta-cell precursors and immunomodulators would be of high interest.