​                                        ‘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.

Chief investigator:    Prof Mohammed Abu Hilal
Study Coordinator:  Christoph Kummerli
 
Without a spleen we run the risk of developing overwhelming (life-threatening) infections. Often the spleen is removed following pancreatic surgery and as a result lifelong antibiotics and vaccines are prescribed for patients. 

The aim of this study is to perform re-implant part of the spleen (26% of the original volume) in patients who lose their spleen during pancreas surgery and to investigate the performance of the re-implanted spleen with the antibody response to a Salmonella Typhi vaccine. Antibodies are measured before and after vaccination and a difference in antibodies will tell us how well the implanted spleen is performing.  In the long-term, the aim is to preserve the splenic function.

Patients are divided into three groups: spleen-preservation surgery (control group), splenectomy with splenic implantation (intervention group) and without (control group).

Since commencing the project the following centres have been recruiting appropriate patients:

• University Hospital Southampton, UK 
• Virginia Mason, Seattle, USA
• Poliambulanza, Brescia, Italy
• Birmingham University Hospital, UK
• Morriston Hospital, Swansea, UK

A further centre is in the process of setting up at the Miami Cancer Institute at Baptist Health, Miami, USA.

At the time of writing this report 28 patients have been recruited.  Progress in reaching the target of 45 by October 2020 might be impacted by the Covid-19 pandemic.  However, patients with low malignant cancer can now also be included. 
 
A further update report will be provided in 6 months time.  LAPR&D has committed to maintain its funding of this project despite the challenges presented by Covid-19.
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Picture: Christoph Kummerli (Study Coordinator) being presented with the research grant by Charity Chairman Kelvin Davis

LAPR&D pledge to continue to support vital research during the Covid-19 Pandemic  – LAPR&D has three important research projects underway at the present time (please see the Research section of this website for details).  However, they have all been impacted to differing degrees by the worldwide Covid-19 pandemic.  In some cases this is as a result of an inability to recruit patients due to non-critical operations being postponed or as a result of our researchers being utilised to support the fight against Coronavirus.  However, LAPR&D Chairman Kelvin Davis committed charity funds to the future of these projects and said “these projects remain vital to improve the life chances of those facing the challenges presented by liver and pancreatic cancer.  I have agreed that funding already promised towards these projects remains in place and project timescales will be extended where projects have been impacted by the Covid-19 pandemic. 

We wish all our researchers and charity supporters well over these difficult months ahead.  Our fundraising events will be postponed but we hope to be back with our exciting events once it is safe to do so.  In the meantime if you wish to donate please use the “donate” buttons on either our Facebook page or website.”

Almost eighty guests and supporters attended the annual curry night at Kuti's in Southampton (www.kutis.co.uk) last Monday to celebrate the work of the charity.  Guests enjoyed the superb food and wonderful hospitality of Kuti and his team.  "It is no wonder this venue won the Tiffin Cup,  making Kuti's the Best Indian Restaurant in the UK, they certainly put on a great show for our supporters" said Charity Patron, Kelvin Davis.   This contributed to our valued followers having a most memorable evening, judging by the feedback received from guests.  

The opportunity was also taken by Christoph  Kummerli (pictured with Charity Patron Kelvin Davis) to introduce the latest research project to be funded by LAPR&D.  As well as supporting Christoph and his fellow research student Alma Moekotte for 2 years,  the charity is also funding the RESTORE trial.  The trail will test the viability of restoring the spleen function when it is removed during surgery, often as part of pancreatic cancer surgery.  Christoph explained "the spleen performs an important function in purifying blood and is a key functions in immune response to fight infection.  When a patient loses their spleen during surgery, they require regular vaccination and a lifelong commitment to antibiotics to replicate the important function of the spleen.  This research project will test the viability of reimplanting part of the healthy spleen into a pocket that we form inside the patient to see if it will recover and begin to reperform it's functions". 

The trail will take 6-12 months and will be supervised by Professor Primrose at Southampton University Hospital with Professor Hilal performing the role of Chief Investigator. 

Guests also bought charity Christmas cards and lapel badges to contribute towards a superb £1,755 raised on the evening and further volunteers came forward to assist the charity with their future work.

​A further grant application to extend the liver stem cell research programme already supported by LAPR&D is also under consideration.

Our joint founder Professor Mo Abu Hilal performed his last operation in Southampton on 30 September - a liver resection - before taking up a new post at the Poliambulanza Hospital in Brescia, Italy.  Mo has been instrumental in guiding the charity's research  programme and will continue to provide input as an Honorary member.  The work of the charity will continue under the professional guidance of Dr. Nadeem Tehami who we will introduce to you shortly.  In the meantime we wish Mo every success in his new post and thank him for his support and kindness over many years since the charity's formation in 2012.

​Following the extension of funding for this project by LAPRD last year, Mogib Khedr (LAP researcher pictured) has conducted 172 experiments involving human samples and 78 experiments involving cell lines and liver stem cells.

Mogib is has made important strides using a novel method to grow liver and liver stem cells. This unique method means that these cells can now live and function for up to two weeks in the laboratory. The technique allows the generation of mature liver cells (from stem cells) and this will enable researchers to test if the cells are able to tolerate anti-cancer treatment. This research has successfully optimised conditions suitable for stem cells, liver cells and most recently cancer cells. Preliminary results of this interesting practice have been recognised and were presented to the national conference ‘The British Association for the Study of the Liver (BASL) Annual Meeting, York 2018’.

In addition to the research above the team have also been working with the Faculty of Engineering at Southampton University to use ultrasonic waves to create a complex structure of cells. The aim of this is to replicate cells normal conditions in the human body where cells would interact with each other and be in an organised 3D form.  This technique allows us to imitate tumours seen in the human body and test anti-cancer drugs more accurately.  This has been published and the full article can be found https://doi.org/10.1063/1.5082603.

Exciting future research will look at patients with liver tumours undergoing surgical intervention. The research will study the role of stem cells during liver regeneration.

This current research project is fully funded by LAPRD until 2020.

Following the overwhelming feedback from our Facebook poll in November 2018 that the charity should focus on its research programme associated with improving the life chances of Liver and Pancreatic cancer patients the charity has approved a "ten point plan" to deliver the programme.  The plan includes:
 
1.  Better communicate the uniqueness of our charity and  projects nationwide - this will focus on our research  programme;

2. Leverage Southampton Hospital Charities for media  relations and communications of key messages and results - initially this will involve meeting with hospital charity representatives to identify where they can provide support;

3.  Improve corporate leaflet – issue new 3 folder flyer;

4.  Target large local companies for corporate sponsorship;

5.  Establish relationships with organisations that raise  awareness - add links to our social media and website;  

6.   Split the role of patron and chairperson; 

7.   Nominate event manager for fund raising activities and set-up formal events bank account and email address;

8.    Strengthen Committee and expand number of volunteers supporters beyond existing   group;

9.    Identify person who manages /updates website, issues emails and social media and

10. Implement monthly donor scheme

If you are able to offer any support for any of these roles or objectives then please do get in touch.  We will keep you updated on progress with these actions during the year.

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A huge thank you to Tina Hiscox who raised £6962 at the last charity lunch, held alongside the Southampton Boat Show at the Grand Café Southampton on 20th September.  The event included games of bingo, superb auction prizes as well as a wonderful lunch. Our thanks also go out to the Grand Café Southampton team (www.grand-cafe.co.uk) for their support and Jenny Slowen the Bingo caller and compere for the day.

Tina is already planning the next event which will be Lunch with Elvis on March 28th 2019 - watch this space for details but in the meantime save the date!