Here we present a non-genetic method to generate human autologous liver spheroids using mononuclear cells isolated from steady-state peripheral blood.
Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to their nature to cluster in the culture dishes with low or no adhesive characteristics, they form aggregates of multiple liver cells that are called human liver spheroids. The forming of 3D liver spheroids relies on the natural tendency of hepatic cells to aggregate in the absence of an adhesive substrate. These 3D structures possess better physiological responses than cells, which are closer to an in vivo environment. Using 3D hepatocyte cultures has numerous advantages when compared with classical two-dimensional (2D) cultures, including a more biologically relevant microenvironment, architectural morphology that reassembles natural organs as well as a better prediction regarding disease state and in vivo-like responses to drugs. Various sources can be used to generate spheroids, like primary liver tissue or immortalized cell lines. The 3D liver tissue can also be engineered by using human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) to derive hepatocytes. We have obtained human liver spheroids using blood-derived pluripotent stem cells (BD-PSCs) generated from unmanipulated peripheral blood by activation of human membrane-bound GPI-linked protein and differentiated to human hepatocytes. The BD-PSCs-derived human liver cells and human liver spheroids were analyzed by light microscopy and immunophenotyping using human hepatocyte markers.
In recent years three-dimensional (3D) spheroid culture systems have become an important tool to study various areas of cancer research, drug discovery, and toxicology. Such cultures raise great interest because they bridge the gap between two-dimensional (2D) cell culture monolayers and complex organs1.
In the absence of an adhesive surface, compared to the 2D cell culture, the formation of spheroids is based on the natural affinity of these cells to cluster in 3D form. These cells organize themselves into groups consisting of one or more types of mature cells. Free of foreign materials, these cells interact with each other like in their original microenvironment. The cells in 3D culture are much closer and have a proper orientation toward each other, with higher extracellular matrix production than 2D cultures, and constitute a close to natural environment 2.
Animal models have been used for a long time to study human biology and diseases3. In this regard, there are intrinsic differences between humans and animals, which makes these models not entirely suitable for extrapolative studies. 3D culture spheroids and organoids represent a promising tool to study tissue-like architecture, interaction, and crosstalk between different cell types that occur in vivo and can contribute to reducing or even replacing animal models. They are of particular interest for studying the pathogenesis of liver diseases as well as drug screening platforms4.
3D spheroid culture is of particular importance for cancer research as it can eliminate the discontinuity between the cells and their environment by reducing the need for trypsinization or collagenase treatment needed for preparing the tumor cell monolayers for 2D cultures. Tumor spheroids enable the study of how the normal versus malignant cells receive and respond to signals from their surroundings5 and are an important part of tumor biology studies.
Compared to the monolayer, 3D cultures consisting of various cell types resemble tumor tissues in their structural and functional properties and therefore are suitable for studying metastasis and invasion of tumor cells. That is why such spheroid models are contributing to accelerating cancer research6.
Spheroids are also helping to develop the technology to create human organoids because tissue and organ biology are very challenging to study, particularly in humans. Progress in stem cell culture makes it possible to develop 3D cultures like organoids consisting of stem cells and tissue progenitors as well as different types of mature (tissue) cells from an organ with some functional characteristics like a real organ that can be used to model organ development, diseases, but they also can be considered useful in regenerative medicine7.
Primary human hepatocytes are usually used for studying in vitro biology of human hepatocytes, liver function, and drug-induced toxicity. Cultures of human hepatocytes have two main drawbacks, firstly, the limited availability of primary tissue like human hepatocytes, and secondly, the tendency of hepatocytes to rapidly dedifferentiate in 2D culture thereby losing their specific hepatocyte function8. 3D hepatic cultures are superior in this regard and have recently been made from differentiated human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs)9. Bioengineered hepatic 3D spheroids are of particular interest to study development, toxicity, genetic and infectious diseases of the liver, as well as in drug discovery for the treatment of liver diseases10. Lastly, they also have the potential to be used clinically, knowing that acute liver diseases have a mortality rate of nearly 80%, bio-artificial liver and/or hepatic spheroids could potentially rescue these patients by providing partial liver function until a suitable donor can be found11.
We have established a protocol for the generation of human hepatic spheroids using blood-derived pluripotent stem cells (BD-PSCs) to prepare differently sized spheroids containing 4000 to 1 x 106 cells and analyzed them by means of light microscopy and immunofluorescence. We also tested the capability of hepatocyte-specific function, assessing the expression of cytochrome P450 3A4 (CYP3A4) and 2E1 (CYP2E1) enzymes that belong to the cytochrome P450 family that have important roles in cellular and drug metabolism through the process of detoxification12.
Ethical approval was obtained (ACA CELL Biotech GmbH/25b-5482.2-64-1) for performing these experiments and informed consent was signed by all donors before blood extraction in compliance with institutional guidelines.
1. Preparation of mononuclear cells (MNCs) from human peripheral blood (PB)
2. Dedifferentiation of MNCs upon activation with human GPI-anchored glycoprotein
3. Sorting of newly generated dedifferentiated cells
4. Preparation of glass coverslips for the generation of human hepatocytes
5. Coating cell culture plates with biolaminin for 2D hepatic differentiation of BD-PSCs
6. Preparation of hepatocyte differentiation media
7. Culturing hepatic cells differentiated from BD-PSCs
8. 3D spheroid hepatic differentiation
9. Immunofluorescence analysis of newly generated 2D liver cell cultures
10. Live staining of newly formed liver spheroids
11. Examination of spheroids using a fluorescence microscope
We successfully differentiated human BD-PSCs into endoderm/hepatic progenitor cells and hepatocytes by applying a two-step protocol. Morphological changes during the hepatic differentiation process are shown in Figure 1. BD-PSCs differentiate into hepatocytes going through three different stages. The first stage represents the differentiation into endodermal cells L4, the second, differentiation to hepatic progenitor cells (hepatoblast) L8, exhibiting a typical polygonal morphology, and the third, the maturation to hepatocytes L15-L24.
Immunofluorescence analysis was performed to confirm the hepatic differentiation of BD-PSCs as presented in Figure 2. Strong expression of endoderm/human liver progenitor marker, like alpha-fetoprotein (AFP), a major plasma protein in fetal serum whose concentration is very low in adult organisms and is therefore considered as a marker for hepatocytes' precursor15 and transthyretin (TTR), a major thyroid-hormone binding protein involved in transporting thyroxine from the bloodstream to the brain16 are found in the cells at the first stage of the hepatic differentiation process at L4 to L8. However, their expression decreases at L15, while the expression of albumin (ALB), the most abundant plasma protein produced mainly by the liver and utterly critical for hepatic differentiation, as well as hepatocyte nuclear factor 4 alpha (HNF-4α), a hepatocytes transcription factor that is involved in the expression of liver-specific genes17 appears firstly at L4, rises throughout the differentiation time L4-L15 reaching a strong and stable expression during the maturation time L15-L24.
Cytokeratin 18 (CK18) is a cytoskeletal protein, one of the major components of intermediate filament expressed in the liver18. The results show that, as expected, CK18 expression correlates with mature hepatocytes (L15-L24), and it is not expressed in hepatocyte progenitor cells.
The well-defined protocol for hepatocyte differentiation in 2D cultures enables the engineering of hepatic 3D cultures starting with BD-PSCs.
We demonstrate here that spontaneous aggregation of these cells in low attachment plates containing hepatocyte induction/maturation medium initiates spheroid formation. The growth track was followed by imaging cells at L2, L4, and L7. (Figure 3A) There is a consistent correlation between spheroid volume and the variable number of cells, as presented in Figure 3B.
The liver is an organ in which most of the drugs in the human body get metabolized. Cytochrome P450 is a superfamily of enzymes (monooxygenases) that are of pivotal importance in the processes of drug and cellular metabolism, detoxification of xenobiotics, and homeostasis. To assess the potential functional activity of BD-PSCs derived hepatic spheroids, we analyzed the expression of drug-metabolizing enzymes like CYP3A4 and CYP2E1, members of CYP3 and CYP2 families19.
Most of the drugs that are used today including codeine, cyclosporin A, erythromycin, acetaminophen, and diazepam as well as many steroids and carcinogens, are metabolized due to the activity of CY3A4 enzyme20. CYP2E1 is involved in the metabolism of endogenous substrates like ethylene glycol, benzene, carbon tetrachloride, and particularly the most important highly mutagenic compound like nitrosamine21.
The spheroids that are formed and differentiated according to the protocol at D14, live stained with antibodies to these two enzymes, reveal the potential hepatic functional activity of BD-PSCs derived spheroids (Figure 4).
Figure 1: Differentiation of BD-PSCs to hepatic-like cells. Representative micrographs of morphological changes throughout hepatic differentiation of BD-PSCs showing endodermal L4, or polygonal shape L8 morphology finally reaching maturation state at L15 to L24. Scale bars: upper row 50 µm, lower row 20 µm. Please click here to view a larger version of this figure.
Figure 2: Immunofluorescence analysis of BD-PSCs re-differentiation towards hepatic cells. Endoderm/hepatocytes progenitor and hepatocytes specific markers are expressed during liver differentiation of BD-PSCs in 2D cultures. On days L4 to L8, micrographs show decreased expression of endoderm/hepatic progenitor AFP and TTR while their expression disappeared from L8-L24. Expression of hepatocytes ALB and HNFα markers arise at L4 and increase during maturation, whereas the expression of CK18 appeared first at L15, reaching the maximum at L24. Scale bars for graphs L4-L15: 50 µm and for L24: 20 µm. Control is presented in Supplementary Figure 1. Please click here to view a larger version of this figure.
Figure 3: Formation of 3D spheroids upon hepatic differentiation of BD-PSCs. (A) Variable cell numbers of BD-PSCs starting with 1 x 106 to 4000 cells were seeded into low attachment plates, and differentiation was performed according to the two-stage procedure as stated in the Protocol. The generation of 3D human liver spheroids was imaged at different time points, shown are representative phase contrast images at each time during the culture time period. Scale bar: 200 µm. (B) Diameters of at least 4 hepatic spheroids for each size were measured at L4 using microscope imaging software and volumes were calculated. Error bars show standard deviation. Please click here to view a larger version of this figure.
Figure 4: Hepatocyte functional markers are expressed in BD-PSCs-derived liver spheroids. BD-PSCs were differentiated into hepatocytes. Direct immunofluorescence analysis was performed on live cells at L14 using antibodies to ALB, AFP, CK18, and CYP2E1 and CYP3A4, members of the cytochrome P450 family. Scale bar: 200 µm. Please click here to view a larger version of this figure.
Supplementary Figure 1: Negative control for immunofluorescence analysis of BD-PSCs re-differentiation towards hepatic cells. Endoderm/hepatocytes progenitor and hepatocytes specific markers are expressed during liver differentiation of BD-PSCs in 2D cultures. Scale bar: 100 µm. Please click here to download this File.
The liver is a major organ in the human body with many essential biological functions, such as the detoxification of metabolites. Due to severe liver failures like cirrhosis and/or viral hepatitis, there are nearly 2 million deaths per year worldwide. Liver transplantations rank second in solid organ transplantations worldwide, but only about 10% of the current need is met22.
Primary human hepatocytes (PHH) are often used to study liver toxicity. These cells can be maintained in culture for a short time retaining their specific functions. Also, the number of cells available from a single donor is limited, in addition, these cells cannot be expanded in the culture therefore, the shortage of donor PHH remains the main obstacle for hepatotoxicity studies. PSCs represent a renewal source of human tissues and can be used for the generation of 3D hepatic cultures11.
Liver 3D culture systems show multiple advantages when compared to 2D. Shorter differentiation time and accurate mimicking of in vivo processes enable for more precise studies on drug-induced toxicity, better prediction of liver liability, and are more cost-effective23. The liver spheroid cultures due to their autologous feature could be a great advantage over primary human hepatocytes (PHH) circumventing the disadvantages related to their use and may become a gold standard for application in testing drug toxicity and has a potential future application in regenerative medicine.
We have demonstrated here that BD-PSCs generated from steady-state peripheral blood can be successfully differentiated into endodermal/hepatocyte progenitors/mature hepatocytes with steady albumin secretion and phenotypic stability expressing hepatocyte markers. Moreover, the engineered 3D human hepatocyte spheroid cultures demonstrate the potential functional activity by expressing the enzymes that belong to cytochrome P450, like CYP3A4 and CYP2E1.
The most important step in the protocol is to obtain good quality and number of fresh human MNCs for the reprogramming process. The use of frozen MNCs results in a reduced number of reprogrammed cells.
We have engineered human liver spheroids using activated PBMNCs cultures with and without applying immunomagnetic sorting that separates reprogrammed cells from mature blood cells. The slight difference in using these two methods relies on the higher density of 3D structure when using purified reprogrammed cells. The expression of hepatocyte markers remains consistent in both cell culture preparations.
Due to the limited availability of PHH, the method potentially represents the biologically relevant closest alternative to autologous fresh hepatocytes to study hepatic function in vitro, like xenobiotic metabolisms and liver toxicity, host-pathogen intervention, and cell biology in general. The possibility of using BD-PSCs in regenerative medicine while being autologous and non-teratogenic is the subject matter of further studies in our laboratory.
The authors have nothing to disclose.
The authors are especially grateful for the technical assistance provided by Oksana and John Greenacre. This work was supported by ACA CELL Biotech GmbH Heidelberg, Germany.
Albumin antibody | Sigma-Aldrich | SAB3500217 | produced in chicken |
Albumin Fraction V | Carl Roth GmbH+Co. KG | T8444.4 | |
Alpha-1 Fetoprotein | Proteintech Germany GmbH | 14550-1-AP | rabbit polyclonal IgG |
Biolaminin 111 LN | BioLamina | LN111-02 | human recombinant |
CD45 MicroBeads | Miltenyi | 130-045-801 | nano-sized magnetic beads |
Cell Strainer | pluriSelect | 43-10040-40 | |
CellSens | Olympus | imaging software | |
Centrifuge tubes 50 mL | Greiner Bio-One | 210270 | |
CEROplate 96 well | OLS OMNI Life Science | 2800-109-96 | |
CKX53 | Olympus | ||
Commercially available detergent | Procter & Gamble | nonionic detergent | |
CYP2E1-specific antibody | Proteintech Germany GmbH | 19937-1-AP | rabbit polyclonal antibody IgG |
CYP3A4 | Proteintech Germany GmbH | 67110-1-lg | mouse monoclonal antibody IgG1 |
Cytokeratin 18 | DakoCytomation | M7010 | mouse monoclonal antibody IgG1 |
DMSO | Sigma-Aldrich | D8418-50ML | |
DPBS | Thermo Fisher Scientific | 14040091 | |
FBS | Merck Millipore | S0115/1030B | Discontinued. Available under: TMS-013-B |
Glass cover slips 14 mm | R. Langenbrinck | 01-0014/1 | |
GlutaMax 100x Gibco | Thermo Fisher Scientific | 35050038 | L-glutamine |
Glutaraldehyde 25% | Sigma-Aldrich | G588.2-50ML | |
Goat anti-mouse IgG Cy3 | Antibodies online | ABIN1673767 | polyclonal |
Goat anti-mouse IgG DyLight 488 | Antibodies online | ABIN1889284 | polyclonal |
Goat anti-rabbit IgG Alexa Fluor 488 | Life Technologies | A-11008 | |
HCl | Sigma-Aldrich | 30721-1LGL | |
HepatoZYME-SFM | Thermo Fisher Scientific | 17705021 | hepatocyte maturation medium |
HGF | Thermo Fisher Scientific | PHG0324 | human recombinant |
HNF4α antibody | Sigma-Aldrich | ZRB1457-25UL | clone 4C19 ZooMAb Rbmono |
Hydrocortisone 21-hemisuccinate (sodium salt) | Biomol | Cay18226-100 | |
Knock out Serum Replacement – Multi Species Gibco | Fisher Scientific | A3181501 | KSR |
KnockOut DMEM/F-12 | Thermo Fisher Scientific | 12660012 | Discontinued. Available under Catalog No. 10-828-010 |
MACS Buffer | Miltenyi | 130-091-221 | |
MACS MultiStand | Miltenyi | 130-042-303 | magnetic stand |
MEM NEAA 100x Gibco | Thermo Fisher Scientific | 11140035 | |
Mercaptoethanol | Thermo Fisher Scientific | 31350010 | 50mM |
MiniMACS columns | Miltenyi | 130-042-201 | |
Nunclon Multidishes | Sigma-Aldrich | D6789 | 4 well plates |
Oncostatin M | Thermo Fisher Scientific | PHC5015 | human recombinant |
Paraformaldehyde | Sigma-Aldrich | 158127 | |
PBS sterile | Carl Roth GmbH+Co. KG | 9143.2 | |
Penicillin/Streptomycin | Biochrom GmbH | A2213 | 10000 U/ml |
PS 15ml tubes sterile | Greiner Bio-One | 188171 | |
Rabbit anti-chicken IgG Texas red | Antibodies online | ABIN637943 | |
Roti Cell Iscoves MDM | Carl Roth GmbH+Co. KG | 9033.1 | |
Roti Mount FluorCare DAPI | Carl Roth GmbH+Co. KG | HP20.1 | |
Roti Sep 1077 human | Carl Roth GmbH+Co. KG | 0642.2 | |
Transthyretin antibody | Sigma-Aldrich | SAB3500378 | produced in chicken |
Triton X-100 | Thermo Fisher Scientific | HFH10 | 1% |