Remodeling of Glycosaminoglycans During Differentiation of Adult Human Bone Mesenchymal Stromal Cells Toward Hepatocytes

Abstract

There is a critical need to generate functional hepatocytes to aid in liver repair and regeneration upon availability of a renewable, and potentially personalized, source of human hepatocytes (hHEP). Currently, the vast majority of primary hHEP are obtained from human tissue through cadavers. Recent advances in stem cell differentiation have opened up the possibility to obtain fully functional hepatocytes from embryonic or induced pluripotent stem cells, or adult stem cells. With respect to the latter, human bone marrow mesenchymal stromal cells (hBMSCs) can serve as a source of autogenetic and allogenic multipotent stem cells for liver repair and regeneration. A major aspect of hBMSC differentiation is the extracellular matrix (ECM) composition and, in particular, the role of glycosaminoglycans (GAGs) in the differentiation process. In this study, we examine the influence of four distinct culture conditions/protocols (T1-T4) on GAG composition and hepatic markers. α-Fetoprotein and hepatocyte nuclear factor-4α were expressed continually over 21 days of differentiation, as indicated by real time quantitative PCR analysis, while albumin (ALB) expression did not begin until day 21. Hepatocyte growth factor (HGF) appears to be more effective than activin A in promoting hepatic-like cells through the mesenchymal-epithelial transition, perhaps due to the former binding to the HGF receptor to form a unique complex that diversifies the biological functions of HGF. Of the four protocols tested, uniform hepatocyte-like morphological changes, ALB secretion, and glycogen storage were found to be highest with protocol T2, which involves both early- and late-stage combinations of growth factors. The total GAG profile of the hBMSC ECM is rich in heparan sulfate (HS) and hyaluronan, both of which fluctuate during differentiation. The GAG profile of primary hHEP showed an HS-rich ECM, and thus, it may be possible to guide hBMSC differentiation to more mature hepatocytes by controlling the GAG profile expressed by differentiating cells.

Keywords: GAG temporal remodeling; adult mesenchymal stromal cells; chondroitin sulfate; functional hepatocytes; heparan sulfate; hyaluronan.

FIG. 1.

hBMSC differentiation protocols examined. Top is a diagram representing the duration and critical time point (dashed lines) during growth, differentiation, and maturation of a sequential differentiation program. Combo 1, 2, and 3 represent combinations of different growth factors, chemicals, and media additives based on specific protocols. Bottom table describes specific components and protocols for hBMSC differentiation. hBMSC, human bone marrow mesenchymal stromal cell.

FIG. 2.

Light microscopic analysis of hBMSC upon differentiation toward hepatocyte-like cells on day 21. Arrows indicate cells with spindle-like morphology. Dashed circles represent rounded morphology. 4 × magnification (scale bar 200 μm). Color images are available online.

FIG. 3.

Effect of differentiation protocols on key hepatic markers at different time points. Fold change in gene expression of ALB, AFP, and HNF was analyzed by qRT-PCR. The values of 2^−ΔΔCT were calculated by normalizing to those of undifferentiated hBMSCs (CTR). The data are represented as averages ± SD, n = 3. ALB expression on day 21 was compared to that of human primary hepatocytes (dashed box at day 21). * and ^# indicate statistically significant changes of P < 0.05 and P < 0.003, respectively. AFP, α-fetoprotein; ALB, albumin; CTR, control; hHep, human hepatocytes; HNF, hepatocyte nuclear factor; qRT-PCR, real time-quantitative polymerase chain reaction; SD, standard deviation.

FIG. 4.

ALB secretion at 21 days of differentiation. hBMSCs exposed to activin A resulted in significantly lower ALB levels than direct methods (protocols T2, T3, and T4). Data represented as average ± SD (n = 3). An asterisk (*) represents significance with P < 0.05.

FIG. 5.

Glycogen storage capacity of hBMSCs at 21 days of differentiation. Top row: hBMSCs exposed to 25, 50, and 100 ng/mL of activin A. Bottom row: hBMSCs directly differentiated toward hepatocytes without actvin A. Glycogen stains magenta color. Original magnification of 4 × (scale bar 200 μm). Color images are available online.

FIG. 6.

Cytochromes P450 enzyme activity at 21 days of hBMSC differentiation. CYP450 activity was measured 48 h after induction with 100 μM omeprazole for CYP1A2 and 25 μM rifampicin for CYP2C9 and CYP3A4. Undifferentiated hBMSCs (CTR) represent the negative control and primary hHep represent the positive control. Data represented as average ± SD (n = 3).

FIG. 7.

LC-MS quantification of total glycosaminoglycans from cells collected on days 3, 7, and 21 of hBMSC differentiation. Changes are compared to undifferentiated hBMSCs (CTR) at each time point. Data presented as average ± SD (n = 3). * and ^# indicate statistically significant changes of P < 0.05 and P < 0.003, respectively. LC-MS, liquid chromatography–mass spectrometry.

FIG. 8.

Schematic depiction of the various HS and CS disaccharides. CS, chondroitin sulfate; HS, heparan sulfate. Color images are available online.

FIG. 9.

LC-MS analysis of percent abundance of individual disaccharides from cells collected on days 3, 7, and 21 of hBMSC differentiation. (A) HS and (B) CS. Data are compared to the undifferentiated hBMSCs (CTR) at each time point. Data presented as average ± SD (n = 3). * and ^# indicate statistically significant changes of P < 0.05 and P < 0.003, respectively. Color images are available online.