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.

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.

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.

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.

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.

Cytomegalovirus-associated superior mesenteric vein thrombosis treated with systemic and in-situ thrombolysis.

A 56-year-old patient, first diagnosed with an acute cytomegalovirus infection, presented with progressive abdominal pain because of a superior mesenteric vein thrombosis for which he was treated with systemic thrombolysis and heparin in continuous infusion. As this therapy did not have the intended success after 5 days, an interventional radiological procedure was performed with local thrombolysis in the superior mesenteric artery resulting in recanalisation of the vein. Oral anticoagulation was initiated and continued for a period of 6 months. Mesenteric venous thrombosis is a relatively uncommon cause of mesenteric ischemia that can be associated with severe morbidity and significant mortality. With noninvasive techniques, it is possible to establish a diagnosis in the majority of the cases. The importance of an early diagnosis and therapy - not only with anticoagulation, but also thrombolysis in selected cases - is shown with this case and review of the literature.

Comparative transcriptome analysis of embryonic and adult stem cells with extended and limited differentiation capacity.

BACKGROUND: Recently, several populations of postnatal stem cells, such as multipotent adult progenitor cells (MAPCs), have been described that have broader differentiation ability than classical adult stem cells. Here we compare the transcriptome of pluripotent embryonic stem cells (ESCs), MAPCs, and lineage-restricted mesenchymal stem cells (MSCs) to determine their relationship. RESULTS: Applying principal component analysis, non-negative matrix factorization and k-means clustering algorithms to the gene-expression data, we identified a unique gene-expression profile for MAPCs. Apart from the ESC-specific transcription factor Oct4 and other ESC transcripts, some of them associated with maintaining ESC pluripotency, MAPCs also express transcripts characteristic of early endoderm and mesoderm. MAPCs do not, however, express Nanog or Sox2, two other key transcription factors involved in maintaining ESC properties. This unique molecular signature was seen irrespective of the microarray platform used and was very similar for both mouse and rat MAPCs. As MSC-like cells isolated under MAPC conditions are virtually identical to MSCs, and MSCs cultured in MAPC conditions do not upregulate MAPC-expressed transcripts, the MAPC signature is cell-type specific and not merely the result of differing culture conditions. CONCLUSION: Multivariate analysis techniques clustered stem cells on the basis of their expressed gene profile, and the genes determining this clustering reflected the stem cells' differentiation potential in vitro. This comparative transcriptome analysis should significantly aid the isolation and culture of MAPCs and MAPC-like cells, and form the basis for studies to gain insights into genes that confer on these cells their greater developmental potency.

7. 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.

Safety and efficacy of weekly docetaxel in frail and/or elderly patients with metastatic breast cancer: a phase II study.

This phase II study was designed to evaluate the safety and efficacy of weekly docetaxel (36 mg/m) for the treatment of metastatic breast cancer in 47 frail and/or elderly patients who were ineligible for the standard 3-weekly docetaxel (100 mg/m) regimen. Reasons for ineligibility to the latter were age > or = 70 years (10 patients), poor hematological reserves (15 patients), impaired liver function (eight patients), intolerance to previous taxanes administered 3-weekly without demonstrated resistance (five patients) or any combination of these reasons (nine patients). There was a median of two prior chemotherapy regimens and more than 60% had a WHO performance score at baseline of 2-3. A total of 408 weekly administrations were given over a period of 525 weeks (78% of the intended dose intensity) and the median cumulative dose of docetaxel per patient was 278 mg/m. The incidence of serious adverse events was low. Grade 3 neutropenia occurred in six patients and grade 4 in four patients. Of these 10 patients, eight had pre-existing hematological abnormalities and four developed neutropenic fever. Neurotoxicity was mild and grade 3 paraesthesia occurred in one patient. The overall objective response rate in 37 evaluable patients was 30% and responses were observed in all subgroups of patients. We conclude that weekly docetaxel (36 mg/m) is active, safe and well tolerated in heavily pre-treated frail/elderly patients with poor prognostic features, including low performance scores and multiple metastatic sites, who would not be eligible for treatment with the standard 3-weekly regimen.