Cell Expansion During Directed Differentiation of Stem Cells Toward the Hepatic Lineage.

The differentiation of human pluripotent stem cells toward the hepatocyte lineage can potentially provide an unlimited source of functional hepatocytes for transplantation and extracorporeal bioartificial liver applications. It is anticipated that the quantities of cells needed for these applications will be in the order of 109-1010 cells, because of the size of the liver. An ideal differentiation protocol would be to enable directed differentiation to the hepatocyte lineage with simultaneous cell expansion. We introduced a cell expansion stage after the commitment of human embryonic stem cells to the endodermal lineage, to allow for at least an eightfold increase in cell number, with continuation of cell maturation toward the hepatocyte lineage. The progressive changes in the transcriptome were measured by expression array, and the expression dynamics of certain lineage markers was measured by mass cytometry during the differentiation and expansion process. The findings revealed that while cells were expanding they were also capable of progressing in their differentiation toward the hepatocyte lineage. In addition, our transcriptome, protein and functional studies, including albumin secretion, drug-induced CYP450 expression and urea production, all indicated that the hepatocyte-like cells obtained with or without cell expansion are very similar. This method of simultaneous cell expansion and hepatocyte differentiation should facilitate obtaining large quantities of cells for liver cell applications.

Osteogenic Potential of Multipotent Adult Progenitor Cells for Calvaria Bone Regeneration.

Osteogenic cells derived from rat multipotent adult progenitor cells (rMAPCs) were investigated for their potential use in bone regeneration. rMAPCs are adult stem cells derived from bone marrow that have a high proliferation capacity and the differentiation potential to multiple lineages. They may also offer immunomodulatory properties favorable for applications for regenerative medicine. rMAPCs were cultivated as single cells or as 3D aggregates in osteogenic media for up to 38 days, and their differentiation to bone lineage was then assessed by immunostaining of osteocalcin and collagen type I and by mineralization assays. The capability of rMAPCs in facilitating bone regeneration was evaluated in vivo by the direct implantation of multipotent adult progenitor cell (MAPC) aggregates in rat calvarial defects. Bone regeneration was examined radiographically, histologically, and histomorphometrically. Results showed that rMAPCs successfully differentiated into osteogenic lineage by demonstrating mineralized extracellular matrix formation in vitro and induced new bone formation by the effect of rMAPC aggregates in vivo. These outcomes confirm that rMAPCs have a good osteogenic potential and provide insights into rMAPCs as a novel adult stem cell source for bone regeneration.

Hepatic differentiation of human embryonic stem cells on microcarriers.

Translation of stem cell research to industrial and clinical settings mostly requires large quantities of cells, especially those involving large organs such as the liver. A scalable reactor system is desirable to ensure a reliable supply of sufficient quantities of differentiated cells. To increase the culture efficiency in bioreactor system, high surface to volume ratio needs to be achieved. We employed a microcarrier culture system for the expansion of undifferentiated human embryonic stem cells (hESCs) as well as for directed differentiation of these cells to hepatocyte-like cells. Cells in single cell suspension were attached to the bead surface in even distribution and were expanded to 1×10(6)cells/ml within 2 days of hESC culture with maintenance of the level of pluripotency markers. Directed differentiation into hepatocyte-like cells on microcarriers, both in static culture and stirred bioreactors, induced similar levels of hepatocyte-like cell differentiation as observed with cells cultured in conventional tissue culture plates. The cells expressed both immature and mature hepatocyte-lineage genes and proteins such as asialoglycoprotein receptor-1 (ASGPR-1) and albumin. Differentiated cells exhibited functional characteristics such as secretion of albumin and urea, and CYP3A4 activity could be detected. Microcarriers thus offer the potential for large-scale expansion and differentiation of hESCs induced hepatocyte-like cells in a more controllable bioreactor environment.

Stem cells and liver engineering.

Human hepatocytes, suitable for treatment of patients with liver failure, for the creation of bioartificial (BAL) devices, or for studies for toxicity and metabolization studies in the pharmaceutical industry, are in short supply due to the lack of donor organs. Therefore, methods that allow ex vivo expansion of hepatocytes with mature function are being pursued. One cell source, believed to be a possible inexhaustible source of hepatocytes, is pluripotent stem cells (PSCs). However, directed differentiation of PSCs to cells with features of adult hepatocytes is not yet possible. Differentiated progeny remains mixed and PSC progeny does not have a number of the functional features of mature hepatocytes. In this review article, we will address tools being developed that allow for the identification of mature hepatocytes, in a non-invasive manner; to perform lineage tracing of PSC progeny; and novel culture systems being created for the in vitro differentiation of PSCs to hepatocyte like cells, and for the maintenance of primary liver derived hepatocytes or PSC-derived hepatic progeny in culture. As conventional two-dimensional (2D) static culture conditions poorly recapitulate the in vivo cellular environment, we will discuss bioreactor systems for liver tissue engineering, both macro-scale and micro-scale culture systems.

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.