Directing hepatic differentiation of embryonic stem cells with protein microarray-based co-cultures

All figure captions are from the Revzin group authors.

Introduction

In order to treat many degenerative liver diseases, recent research has turned to directed differentiation of embryonic stem cells (ESCs) into hepatocytes, either in vitro or in vivo, due to the shortage of livers for transplantation and other alternative therapies. Unfortunately, the conversion of ESCs into mature hepatocytes has encountered very low efficiencies, making the use of ESCs for liver disease therapies impracticable. However, research groups have experienced successes utilizing in vitro differentiation methods, in which a local microenvironment or niche does not have to be solely engineered; rather, by using mature hepatocytes in co-culture with ESCs, researchers can exploit the natural microenvironment of hepatocytes without having to determine each factor alone. Microenvironmental concerns include secreted cell signals such as growth factors, as well as the extracellular matrix. This co-culture method of differentiating ESCs in the same tissue culture plate as mature hepatocytes has been widely employed. However, the method presents two obvious drawbacks which this paper seeks to remedy: (1) random co-culture of ESCs with primary cells allows infinite and uncontrollable cell-cell interactions and (2) isolation of differentiated ESCs for further analysis proves difficult in a random co-culture. These problems have often been solved with micropatterning, which A.Revzin’s group also proposes in this paper. Printed arrays of collagen and fibronectin were used to localize hepatocytes and ESCs in a known spatial orientation. This method also allowed ESCs to be easily removed via laser catapulting for RT-PCR analysis of hepatocyte-related genes. The group ultimately determined that micropatterning allowed for effective co-culture of ESCs with primary hepatocytes and efficient removal of ESCs for further study.

Results

Revzin’s group micropatterned ECM arrays on glass slides as in Figure 1. The micropatterning technique allowed the researchers to localize cell populations and to strictly control distances between cell populations. The cells were then seeded onto their respective ECM proteins (mouse ESCs on fibronectin, hepatocytes on collagen I).



Fig. 1 Schematic description of the assembly of mESC–hepatic cell co-cultures on protein microarrays. Step 1: columns of fibronectin (blue) and collagen (I) (red) spots are printed onto a silane-modified (gray) glass slide. Step 2: after incubation for 1 h and removal of unattached cells, mESCs remained adherent exclusively on the fibronectin spots. Step 3: hepatic cells (HepG2) were seeded on the same surface 24 h after mESC seeding. Hepatic cells attached on collagen I) spots. Step 4: after cultivation for the desired period of time mESCs were extracted from the surface using laser catapulting and were analyzed with RT-PCR.




As seen in Figure 2, alkaline phosphatase staining was conducted on the micropatterned slides to ensure proper cell seeding, as AP stains stem cells but not hepatocytes. Over time, the cell populations clearly expanded out past the original attachment sites.

Fig. 2 Creating micropatterned co-cultures of mESCs and hepatic cells on protein arrays. (A) Seeding mESCs onto arrays comprised of alternating 300 m diameter spots of fibronectin and collagen (I) resulted in selective attachment of stem cells on fibronectin. FITC-labeled collagen (I) was used to highlight that collagen islands were free of stem cells and were available for hepatocyte attachment. (B) Staining co-cultures for alkaline phosphatase—a stem cell marker—revealed a strong signal from the stem cell-containing spot (right) and no signal from the hepatic cell cluster (left). (C) A low magnification (4×) view of the micropatterned co-cultures shows a 14 × 10 array of cell clusters. Clusters of mESCs appear more three dimensional and scatter light, while hepatic cells appear planar and darker. (D–E) Stem cells in mono- or co-cultures were able to expand out of the original attachment sites over time in culture. The images shown here were taken on day 8. Mouse ESCs adherent on fibronectin spots and cultured in hepatic differentiation media (D) were compared to mESC-hepatic cells co-cultures (E). (F) A higher magnification view of the stem cell cluster after 8 d in co-culture shows endodermal-looking cells spreading/migrating out of the cluster.


Figure 3 illustrates the efficiency of the laser catapulting technique employed by Revzin’s group that allowed them to remove a cell cluster independent of another. Laser catapulting offers the ability to analyze and link gene expression of a given cell clump to its location on the slide. Figure 3 also demonstrates the decrease in pluripotent gene expression in ESCs, indicating differentiation. However, the RT-PCR comparisons also indicate some subset of the ESC population remains undifferentiated. The paper states that this perhaps results from the fact that the centrally located ESCs were primarily undifferentiated, while the border cells closest to the hepatocyte co-culture did differentiate. This result indicates the importance of cellular interactions for the induction of hepatocyte cells. Similarly, they tested for expression of genes unique to the three germ layers, discovering the increase of mesodermal genes in differentiated ESCs.

Fig. 3 RT-PCR analysis of pluripotency and germ layer gene expression in stem cell micropatterns. Stem cells were cultured in hepatic differentiation media either alone or together with hepatic cells (HepG2). (A) Mouse ESCs were selectively retrieved from the co-culture using laser catapulting and were immediately used in RT-PCR studies. (B) Expression of pluripotency gene Nanog decreased over time for both mono- and co-cultivated mESCs; however, the co-culture induced a more pronounced and more rapid decrease in Nanog expression. (C) Expression levels of germ layer genes at day 1 and day 8. The endodermal marker, Sox17, was up-regulated, while other germ layer markers remained unchanged or decreased.







Additionally, researchers tested liver-related genes directly, finding that liver-specific gene expression appeared over time in the differentiated ESCs. Figure 4 illustrates the appearance of early hepatic genes. Finally, researchers tested the ESCs for mature hepatocyte markers, as seen in Figure 5. While the mature markers were not present early on, they appeared later in the co-culture stages and at the periphery of the cell population, indicating successful differentiation to hepatocytes and the importance of utilizing mature hepatocytes as a co-culture mechanism.


Fig. 4 Expression of early hepatic phenotype in micropatterned mESC cultures. (A) Expression of Afp, TTR and Alb-genes associated with early hepatic phenotype-occurred earlier and was more pronounced in mESC co-cultures compared to mono-cultures. (B) Immuno-fluorescent staining was used to corroborate the presence of AFP protein in mESC-derived cells after 5 d in culture.









Fig. 5 Expression of genes associated with mature hepatic phenotype. (A) In order to investigate location-specific differences in gene expression, cells were laser-catapulted from the center of the stem cell cluster as well as from the periphery of the cluster (edge). (B) Cells from different locations were catapulted into distinct centrifuge tubes and were analyzed using RT-PCR. These studies revealed considerable heterogeneity in G6p and Ggt gene expression with expression levels being higher at the edge (stem cell–hepatocyte interface) compared to the center of stem cell spot. It should be noted that no G6p and Ggt expression was observed in mono-cultured stem cells.









Critique

Though the cell populations seeded onto the micropatterned slides were localized, they were not immobilized. As seen in Figure 2, many of the populations expanded past their printed ECM borders, perhaps contributing to population mixing. This problem could perhaps be alleviated by immobilizing the cells on the ECM by adding an additional ECM protein. However, the paper states that a silane coating on the slides purposely allowed for the expansion of cell populations, which facilitated cell-cell interactions at the borders. While the paper argues that the populations remained distinct at the borders, cell mixing may still occur and steps could be taken to avoid contamination.

The researchers also utilized a culture media containing insulin for the co-culture incubation. While this addition perhaps assisted the differentiation process, it also somewhat defeats the purpose of co-culture, as the media now helps differentiate ESCs, rather than allowing the hepatocytes to secrete all necessary signals to direct differentiation. Additionally, an ESC population subjected to hepatocyte differentiation media was used as a control to compare media-differentiation and co-culture differentiation. While this method displayed good results (co-culture being more successful in differentiating ESCs), the media used cannot be wholly reliable as not all factors for directed differentiation can be determined.

Finally, this paper admitted problems with RT-PCR results, as the cycle to threshold number was difficult to determine despite multiple experiments. The paper did not state how the cell population was homogenized before starting RT-PCR, so a possibility for future studies would be to re-analyze the differentiated ESCs and control ESCs with a homogenized cell sample.