Karen Pauwelyn

Stem and progenitor cells for liver repopulation: can we standardise the process from bench to bedside?

There has been recent progress in the isolation and characterisation of stem/progenitor cells that may differentiate towards the hepatic lineage. This has raised expectations that therapy of genetic or acquired liver disease might be possible by transplanting stem/progenitor cells or their liver-committed progeny. However, it is currently impossible to determine from the many documented studies which of the stem/progenitor cell populations are the best for therapy of a given disease. This is largely because of the great variability in methods used to characterise cells and their differentiation ability, variability in transplantation models and inconsistent methods to determine the effect of cell grafting in vivo. This manuscript represents a first proposal, created by a group of investigators ranging from basic biologists to clinical hepatologists. It aims to define standardised methods to assess stem/progenitor cells or their hepatic lineage-committed progeny that could be used for cell therapy in liver disease. Furthermore standardisation is suggested both for preclinical animal models to evaluate the ability of such cells to repopulate the liver functionally, and for the ongoing clinical trials using mature hepatocytes. Only when these measures have been put in place will the promise of stem/progenitor-derived hepatocyte-based therapies become reality.

Human Embryonic and Rat Adult Stem Cells with Primitive Endoderm-Like Phenotype Can Be Fated to Definitive Endoderm, and Finally Hepatocyte-Like Cells

Stem cell-derived hepatocytes may be an alternative cell source to treat liver diseases or to be used for pharmacological purposes. We developed a protocol that mimics mammalian liver development, to differentiate cells with pluripotent characteristics to hepatocyte-like cells. The protocol supports the stepwise differentiation of human embryonic stem cells (ESC) to cells with characteristics of primitive streak (PS)/mesendoderm (ME)/definitive endoderm (DE), hepatoblasts, and finally cells with phenotypic and functional characteristics of hepatocytes. Remarkably, the same protocol can also differentiate rat multipotent adult progenitor cells (rMAPCs) to hepatocyte-like cells, even though rMAPC are isolated clonally from cultured rat bone marrow (BM) and have characteristics of primitive endoderm cells. A fraction of rMAPCs can be fated to cells expressing genes consistent with a PS/ME/DE phenotype, preceding the acquisition of phenotypic and functional characteristics of hepatocytes. Although the hepatocyte-like progeny derived from both cell types is mixed, between 10–20% of cells are developmentally consistent with late fetal hepatocytes that have attained synthetic, storage and detoxifying functions near those of adult hepatocytes. This differentiation protocol will be useful for generating hepatocyte-like cells from rodent and human stem cells, and to gain insight into the early stages of liver development.

Directed differentiation of murine-induced pluripotent stem cells to functional hepatocyte-like cells

BACKGROUND & AIMS Induced pluripotent stem (iPS) cells exert phenotypic and functional characteristics of embryonic stem cells even though the gene expression pattern is not completely identical. Therefore, it is important to develop procedures which are specifically oriented to induce iPS cell differentiation. METHODS In this study, we describe the differentiation of mouse iPS cells to hepatocyte-like cells, following a directed differentiation procedure that mimics embryonic and fetal liver development. The sequential differentiation was monitored by real-time PCR, immunostaining, and functional assays. RESULTS By sequential stimulation with cytokines known to play a role in liver development, iPS cells were specified to primitive streak/mesendoderm/definitive endoderm. They were then differentiated into two types of cells: those with hepatoblast features and those with hepatocyte characteristics. Differentiated hepatocyte-like cells showed functional properties of hepatocytes, such as albumin secretion, glycogen storage, urea production, and inducible cytochrome activity. Aside from hepatocyte-like cells, mesodermal cells displaying some characteristics of liver sinusoidal endothelium and stellate cells were also detected. CONCLUSIONS These data demonstrate that a protocol, modeled on embryonic liver development, can induce hepatic differentiation of mouse iPS cells, generating a population of cells with mature hepatic phenotype.

Liver repopulation with bone marrow-derived cells improves the metabolic disorder in the Gunn rat

Background: Reversible ischaemia/reperfusion (I/R) liver injury has been used to induce engraftment and hepatic parenchymal differentiation of exogenous β2-microglubulin−/Thy1+ bone marrow derived cells. Aim: To test the ability of this method of hepatic parenchymal repopulation, theoretically applicable to clinical practice, to correct the metabolic disorder in a rat model of congenital hyperbilirubinaemia. Methods and results: Analysis by confocal laser microscopy of fluorescence labelled cells and by immunohistochemistry for β2-microglubulin, 72 hours after intraportal delivery, showed engraftment of infused cells in liver parenchyma of rats with I/R, but not in control animals with non-injured liver. Transplantation of bone marrow derived cells obtained from GFP-transgenic rats into Lewis rats resulted in the presence of up to 20% of GFP positive hepatocytes in I/R liver lobes after one month. The repopulation rate was proportional to the number of transplanted cells. Infusion of GFP negative bone marrow derived cells into GFP positive transgenic rats resulted in the appearance of GFP negative hepatocytes, suggesting that the main mechanism underlying parenchymal repopulation was differentiation rather than cell fusion. Transplantation of wild type bone marrow derived cells into hyperbilirubinaemic Gunn rats with deficient bilirubin conjugation after I/R damage resulted in 30% decrease in serum bilirubin, the appearance of bilirubin conjugates in bile, and the expression of normal UDP-glucuronyltransferase enzyme evaluated by polymerase chain reaction. Conclusions: I/R injury induced hepatic parenchymal engraftment and differentiation into hepatocyte-like cells of bone marrow derived cells. Transplantation of bone marrow derived cells from non-affected animals resulted in the partial correction of hyperbilirubinaemia in the Gunn rat.

Isolation Procedure and Characterization of Multipotent Adult Progenitor Cells from Rat Bone Marrow

Multipotent adult progenitor cells (MAPCs) are adult stem cells derived from the bone marrow of mouse and rat and were described for the first time in 2002 (Jiang et al., Nature 418:41-49, 2002), and subsequently (Breyer et al., Exp Hematol 34:1596-1601, 2006; Jiang et al., Exp Hematol 30:896-904, 2002; Ulloa-Montoya et al., Genome Biol 8:R163, 2007). The capacity of rodent MAPC to differentiate at the single-cell level into some of the cell types of endoderm, mesoderm, and neuroectoderm germ layer lineages makes them promising candidates for the study of developmental processes. MAPC are isolated using adherent cell cultures and are selected based on morphology after a period of about 8-18 weeks. Here, we describe a step-by-step reproducible method to isolate rat MAPC from fetal and adult bone marrow. We elaborate on several aspects of the isolation protocol including, cell density and medium components, and methods for selecting and obtaining potential MAPC clones and their characterization.

Differentiation of rat multipotent adult progenitor cells to functional hepatocyte-like cells by mimicking embryonic liver development

Differentiation of stem cells to hepatocytes has industrial applications, as well as the potential to develop new therapeutic strategies for liver disease. The protocol described here, sequentially using cytokines that are known to have a role in liver embryonic development, efficiently differentiates rat multipotent adult progenitor cells (rMAPCs) to hepatocyte-like cells by directing them through defined embryonic intermediates, namely, primitive streak/mesendoderm/definitive endoderm, hepatoblast and hepatocyte-like phenotype. After 20 days, the final differentiated multipotent adult progenitor cell progeny is a mixture of cells, comprising cells with the characteristics of hepatoblasts and a smaller cell fraction with the morphological and phenotypical features of mature hepatocytes, as well as other mesodermal cells and some persistent undifferentiated rMAPCs. A detailed functional characterization of the stem cell progeny is also described; this should be used to confirm that differentiated cells display the functional characteristics of mature hepatocytes, including albumin secretion, glycogen storage and several detoxifying functions such as urea production, bilirubin conjugation, glutathione S-transferase activity and cytochrome activity.

Transplantation of Undifferentiated, Bone Marrow‐Derived Stem Cells

Stem cell research has known an enormous development, and cellular transplantation holds great promise for regenerative medicine. However, some aspects, such as the mechanisms underlying stem cell plasticity (cell fusion vs true transdifferentiation) and the functional improvement after stem cell transplantation, are highly debated. Furthermore, the great variability in methodology used by several groups, sometimes leads to confusing, contradicting results. In this chapter, we review a number of studies in this area with an eye on possible technical and other difficulties in interpretation of the obtained results.

MAPC culture conditions support the derivation of cells with nascent hypoblast features from bone marrow and blastocysts

Dear Editor, We previously demonstrated (Jiang et al., 2002) that rodent multipotent adult progenitor cells (MAPC) can self-renew longterm while maintaining multilineage differentiation capacity. Rodent MAPC express a number of pluripotency-related transcription factors (TF) including Oct4 and Rex1 but not Nanog and Sox2, two other TF known to play a significant role in the maintenance of the pluripotency of embryonic stem cells (ESC) (Ulloa-Montoya et al., 2007). However, rodent MAPC express several TF, including Gata4, Gata6, Sox7 and Sox17, typically expressed in the nascent hypoblast of the developing inner cell mass (ICM) (Nichols and Smith, 2011) and in the recently described rat extrambryonic endodermal precursor cells (rXEN-P), which are isolated from blastocyst (Debeb et al., 2009). We derived in 4/12 independent isolations one or more rMAPC lines, by culturing rat BM cells in rMAPC medium (rMAPC isolation scheme, Supplementary Figure S1). After 4 weeks of culture, BM cells were depleted of CD45+ cells and 2–8 weeks later, clusters of refractile and small cells appeared, which became the preponderant cell type within 10 days (Figure 1A). Nearly all cells from the established lines expressed Oct4, Gata4, Gata6, Sox7 and Sox17 transcripts and proteins (Figure 1B and Supplementary Figure S2A and B), as well the surface markers SSEA1 and CD31 (Figure 1C and Supplementary Figure S2C), both markers of the early ICM. Although rMAPC lines express Oct4, previous studies (Lengner et al., 2007) demonstrated that Oct4+ cells cannot be detected in adult mouse tissues and that Oct4 is not required for postnatal tissue homeostasis. Based on Lengner’s findings, we hypothesized that the rMAPC phenotype could be the result of a culture-induced reprogramming. We therefore analysed BM-cultures during 2 independent rMAPC isolations before, during and after the appearance of the refractile and small cells. We could not identify any Oct4+ or SSEA1+/CD31+ cells in more than one million cells analyzed after CD45+ cells depletion (Figure 1C and D, and Supplementary Figure S2D and E), several weeks before the appearance of the refractile cells positive for these markers. RT-qPCR analysis further demonstrated that acquisition of the typical rMAPC morphology was associated with .1000fold increase in expression of Oct4 and the typical hypoblast gene transcripts (Figure 1E and Supplementary Figure S2F). Although some rMAPC lines had karyotypical abnormalities (Supplementary Figure S2G and Table S1), some lines did not, suggesting that the rMAPC phenotype is not induced by a specific translocation, duplication and/or deletion. These studies demonstrate that rMAPC do not exist in BM and that this hypoblast phenotype is acquired upon prolonged in vitro culture. rMAPC may represent a rare event of in vitro reprogramming, resembling what has been observed during spermatogonial stem cell (Guan et al., 2006; Kanatsu-Shinohara et al., 2008; Ko et al., 2009) and epiblast stem cell (Bao et al., 2009) de-differentiation to ESC-like cells, when cultured under ESC conditions. Because the gene expression pattern of rMAPC (Ulloa-Montoya et al., 2007) and rXEN-P cells (Debeb et al., 2009) is highly similar, we next asked whether BM cells were reprogrammed to a hypoblast/extraembryonic progenitor fate. To investigate this, we tested whether rMAPC could be cultured under rXEN-P conditions and vice versa. When established rXEN-P lines were cultured under rMAPC conditions for 1–2 passages, they grew dispersed, acquiring the typical rMAPC morphology (Supplementary Figure S3A). rXEN-P cells became homogeneously Oct4+/Gata4+ and the percentage of SSEA1+ cells increased (Supplementary Figure S3B and E). RT-qPCR revealed that no differences in RNA expression for hypoblast genes could be detected in XEN-P lines, once cultured in MAPC conditions, except for higher levels of Sox17 and lower levels of Tmprss2 (Supplementary Figure S3D). By contrast, when rMAPC were cultured in XEN-P medium on rat embryonic feeders, typical XEN-P colonies were generated, i.e. Oct42/Gata4+ epithelioid cells with a rim of loosely attached small refractile cells that are Oct4+/Gata4+ (Supplementary Figure S3F and G). Moreover, the percentage of SSEA1+ cells decreased (Supplementary Figure S3J); consistently, RT-qPCR revealed a decrease in Sox17 and an increase in Tmprss2 (Supplementary Figure S3I). Cell doubling time of rMAPC or rXEN-P cells cultured in MAPC conditions was slightly faster than in XEN-P conditions (Supplementary Figure S3A and F). Therefore, rMAPC culture conditions supported the feederfree growth of established XEN-P clones in a more homogenous and immature state. To further define the relationship between rMAPC, XEN-P and typical XEN cells, rMAPC and rXEN-P cells were also cultured under standard XEN conditions (Kunath et al., 2005) without exogenous LIF (Supplementary Figure S4A). rXEN-P and rMAPC cells in XEN conditions, formed extraembryonic endodermal colonies with significantly lower proliferation rate (Supplementary Figure S4B and E). Expression of hypoblast gene transcripts doi:10.1093/jmcb/mjs046 Journal of Molecular Cell Biology (2012), 4, 423–426 | 423 Published online August 9, 2012

Culture of Mouse Embryonic Stem Cells with Serum but without Exogenous Growth Factors Is Sufficient to Generate Functional Hepatocyte-Like Cells

Mouse embryonic stem cells (mESC) have been used to study lineage specification in vitro, including towards a hepatocyte-like fate, and such investigations guided lineage differentiation protocols for human (h)ESC. We recently described a four-step protocol to induce hepatocyte-like cells from hESC which also induced hepatocyte-like cell differentiation of mouse induced pluripotent stem cells. As ESC also spontaneously generate hepatocyte-like cells, we here tested whether the growth factors and serum used in this protocol are required to commit mESC and hESC to hepatocyte-like cells. Culture of mESC from two different mouse strains in the absence of serum and growth factors did not induce primitive streak/definitive endoderm genes but induced default differentiation to neuroectoderm on day 6. Although Activin-A and Wnt3 induced primitive streak/definitive endoderm transcripts most robustly in mESC, simple addition of serum also induced these transcripts. Expression of hepatoblast genes occurred earlier when growth factors were used for mESC differentiation. However, further maturation towards functional hepatocyte-like cells was similar in mESC progeny from cultures with serum, irrespective of the addition of growth factors, and irrespective of the mouse strain. This is in contrast to hESC, where growth factors are required for specification towards functional hepatocyte-like cells. Culture of mESC with serum but without growth factors did not induce preferential differentiation towards primitive endoderm or neuroectoderm. Thus, although induction of primitive streak/definitive endoderm specific genes and proteins is more robust when mESC are exposed to a combination of serum and exogenous growth factors, ultimate generation of hepatocyte-like cells from mESC occurs equally well in the presence or absence of exogenous growth factors. The latter is in contrast to what we observed for hESC. These results suggest that differences exist between lineage specific differentiation potential of mESC and hESC, requiring optimization of different protocols for ESC from either species.