​                                        ‘Isolation and expansion of human liver stem cells’

Principal investigators    Prof. Abu Hilal & Prof. Salim Khakoo    University of Southampton

Researcher                            Mogibelrahman Khedr    University of Southampton    

Collaborators                       Prof. Martyn Hill & Dr Peter Glynne-Jones    University of Southampton
                                                      Faculty of Engineering and the Environment

                                                      Dr Jonathan Dawson    University of Southampton    
​                                                      Bone and Joint Research Group, Faculty of Medicine
 
 Acknowledgments:

The research team wish to express their grateful thanks to the ‘Liver & Pancreatic R&D Cancer Charity’ for their funding and great support of the ‘Isolation and expansion of human liver stem cells’ project in University of Southampton.  We are also intensely thankful for the charity committee, charity members and all funders who worked hard to make this work possible. 
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Introduction:

This project started initially with work on laboratory animals and involved isolation and growing of mouse liver cells.  This initial work, reported on elsewhere on the news section of the LAPR&D website, progressed to using human tissues which provide cells in cultures that resemble those that we have in our bodies.  Collectively, we have recruited patients and used collected tissues in 179 experiments.  In addition, we conducted 81 experiments involving cell lines and liver stem cells.
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Objectives:
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        1.    To grow and maintain functioning liver cells outside of the body and to identify a way to grow the cells as three dimensions model.
        2.    To allow for earlier drug experimentation on functioning cultured liver tissues with reduced risks to patients.
        3.    To isolate liver stem cells, grow them in the lab and evolve liver stem cells to mature liver cells with full liver functions.
        4.    In depth characterisation of newly formed cells at the functional and the genetic levels.
        5.    To apply for grants from further funding sources to continue and expand our work.

Ultimately the aim is to be able to grow a human cell outside of the body from the dna of the patient.  Once proven this will enable surgeons to remove the whole of the liver thereby increasing the change of leaving no cancer cells in the patient before replacing the “grown” liver into the patient.

Outcomes:

        1.    Succeeded in making liver cells grow.

We had studied the growth of liver cells in our laboratory and factors which help maintenance and growth of those cells. We made great progress in this area by unveiling the role of Vasoactive-Intestinal Peptide (VIP) the small protein that is normally found in our livers and which support liver cells to grow and proliferate.  These novel results were published in Wiley’s Journal of Cell Proliferation in 2018.  The full medical research article can be found here: https://doi.org/10.1111/cpr.12482
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       2.    Identify a way to grow the cells in three dimensions. 

The second theme involved methods by which we can involve earlier experimentation of new drugs and reduce risk for patients.  Generation of liver tissue in the laboratory which closely resembles the function of our livers was the aim.  We had established a collaboration with the Faculty of Bioengineering at Southampton University to develop an ultrasound device which might enable us to grow liver cells in an environment resembling normal conditions and hence use it for drug screening.  We established a three dimensional (3D) hepatic cancer cell line culture.  The research successfully tested anti-cancer drugs in this model.  This model will provide an easy and safe way for drug toxicity screening and estimation of anti-cancer drug effect.  The results were published in The Journal of Acoustical Society of America in 2019.  The full medical research paper can be found here: https://doi.org/10.1063/1.5082603

In addition, clay could improve the generation of mature liver cells from stem cells.  Several experiments have been performed to explore some of the essential characteristics of the clay nanoparticle culture.  The team have had good success in modifying the clay matrix, as a composite, to permit culture of various cell type including cancer cell lines.  A new clay composite was successfully used as a 3D matrix for hepatocellular carcinoma cell line. The results confirmed the beneficial use of this material in growing liver cells.  This new technique has successfully led to the formation of sphere like cells in less than one week. These spheroids could be used in screening anti-cancer drugs and could mimic the response of cancer tissue present in the human body.  These results will be published soon.

      3.    Successfully isolated stem cells and evolve these stem cells to mature liver cells with full liver functions.
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We were successfully able to isolate progenitor/stem cells (HPCs) in our laboratory.  We were able to characterize these rare cells, grow and maintain them.  In collaboration with Dr J Dawson from the Human Development & Health department at Soiuthampton University, a novel clay gel matrix has been tested.  The results show that material was able to push stem cells to form mature and functioning liver cells which is a promising step forward towards the fabrication of liver tissue in this gel.  The results of this interesting technique were presented to the national conference ‘The British Association for the Study of the Liver (BASL) Annual Meeting, York 2018’.

4.    In depth characterisation of newly formed cells at the functional and the genetic levels.
 
We have initially tested the functions of formed liver cells in clay material.  Although we have approval for an additional fund to perform a detailed analysis of cell functions and genetic characterisation, we are not able to conduct these experiments due to the Covid pandemic.

5.    Applications for grants.
 
Proposals for research grants have been submitted but have not been successful at present.

We have initiated a new collaboration with the ‘Centre of Regenerative Medicine, Edinburgh’, which is a world class centre in the field aiming to use the novel clay material on a wider scale for the cultivation of liver cells.  We have benefited from the experience and reputation of this centre to draft a proposal for a substantial Engineering and Physical Sciences Research Council (ESPRC) research fund.

Experimental summary:

Growth of liver cells in culture is a challenge.  Cells outside our bodies lack support from their neighbouring cells and separate them from chemical messengers which help their viability and growth.  We have therefore tested the addition of growth factors EGF and VIP to our cells in the culture dish.  This addition has a substantial impact on cell viability, growth and functionality.  Cells produced more albumin, metabolised proteins and urea with these factors added.  A beneficial effect has been demonstrated on liver cell genomic machinery which favours their functions and improves their growth. 

However, the culture of liver cells as a flat layer in laboratory flask is far removed from their organisation in our liver.  Therefore, the assembly of cells in a 3D structure is more physiological.  We had studied an ultrasound wave driven device to create a 3D liver cell culture.  Human liver carcinoma cell lines were used as a model for this technology.  The results show that cells in this device were viable and their functions were better than those grown conventionally.  Moreover, these micro-tissues may mimic the shape of tumour mass in patients which is critical for drug treatment.  The model was successful in showing the effect of anti-cancer drugs.  This is promising as a model for drug toxicity screening. 

In addition to ultrasound device, we have tested a clay gel material to grow cells in a 3D environment.  Interestingly, encapsulating cells in clay gel was found to be helpful in maintaining mature liver cells for longer time than conventional methods.  Moreover, liver cells were able to synthesise more albumin and metabolise urea better than when grown as a flat layer of cells.
Isolation and maintenance of human liver stem cell was challenging.  We have tried several methods including speed isolation and magnetic beads sorting, and we were successfully able to isolate a certain population with stem cell marker.  We have tried several forms of culture medium to maintain cells and fully characterise liver stem cells using their genetic signature such as Thy1, CK19 and Claudin 3.  Growing liver stem cells in clay gel was successful in generation of mature liver cells which was able to produce more albumin.  We have found that VIP treatment improves maintenance of our stem cells in culture.

Summary:

In summary, in our laboratory we have established research in the University of Southampton on liver mature and stem cells which will open the gates for further development, progress and research.  LAPR&D hope to be able to be involved in this promising future research when viable to do so.