Metabolic Disease

On Ray

What is your educational background?

I grew up in rural, eastern North Carolina and attended Wake Forest University in Winston-Salem, where I double-majored in biology and French language and civilization, graduating with honors in May 1993. I then spent a little over a year abroad in Grenoble, France studying molecular and cellular biology at the Université Joseph Fourier. There, I was first introduced to developmental biology both in class and in the lab, working as an intern on a project investigating why chickens develop scales on their feet but feathers on their back. This basic question of patterning-how populations of initially equivalent cells later give rise to specific tissues in time and space-captivated me and led me to pursue a Ph.D. in developmental biology under the supervision of Dr. Brigid Hogan at Vanderbilt University Medical Center, Nashville, TN. During my Ph.D., I closely collaborated with Dr. Kirstie Lawson (then at Utrecht) and we showed that the secreted signaling molecule Bmp4 is absolutely required for formation of the mammalian germline-work that has now been cited more than 900 times. My rather unshakeable interest in embryonic development and lineage specification then carried me to Harvard University and the laboratory of Dr. Liz Robertson. There I investigated at the genetic level the contributions of Smad2 and Smad3, two intracellular effectors of the Nodal ligand, to germ layer specification during mouse gastrulation. It was during my postdoc that I began to home in on my now -favorite germ layer, the endoderm, which gives rise to the liver, lung and all-important pancreas.

Who is/are your scientific idols?

John Gurdon for his pioneering work on nuclear transfer and cloning; Rosa Beddington, an incomparable embryologist and molecular geneticist, who identified an extraembryonic signaling centre in the early mouse embryo responsible for the establishment of the anterior-posterior axis; James Thomson, of course, for the isolation of human embryonic stem cells in 1993; and Jennifer Doudna, Emmanuelle Charpentier and Feng Zhang for making gene editing a breeze-the advent of CRISPR/Cas9 has forever changed the world of research.

What's the focus of your current work?

My laboratory is fundamentally interested in elaborating the transcriptional networks governing human pancreatic development. We model in vitro rare forms of human pancreatic agenesis using gene-edited hESC or patient-derived human induced pluripotent cell (hiPSC) lines. Comparative approaches between wild-type and mutant cells have allowed us to identify the downstream transcriptional targets of key regulators of pancreatic development, such as PDX1. From these studies, we have selected several poorly characterized candidates for further validation, which we posit also function like PDX1 in the adult islet to maintain normoglycemia and are thus prone to dysregulation in diabetes. We are also pursuing the isolation of hESC-derived multipotent PDX1+ pancreatic progenitor cell lines that are both stable and scalable. Such an "intermediate" hESC-derived reagent, we argue, might overcome a number of the drawbacks of hESC-based directed differentiation and provides a novel and robust experimental platform for the downstream production of β cells for cell transplantation. Lastly, through a fortuitous collaboration within my home institute we are investigating rare forms of heritable diabetes and have focused our attention on genes potentially involved in the maintenance of β cell homeostasis.

On PSC Research

What do you consider to be the most important advance(s) in stem cell research over the past 5 years, and what advances do you hope the field will achieve in the next 5 years? See More

The obvious (and indisputable) answer to the first part of this question is the advent of the remarkably easy CRISPR/Cas9 technology for gene editing. An easy tie for first place is the ability to generate induced pluripotent stem cells from terminally differentiated cell types grâce à the Yamanaka factors. The next five years will certainly see increasing efforts to generate multiplex organoids in vitro to simulate in vivo organ formation. Mini-brains and mini-guts will be joined by mini- pancreases, mini-livers and mini-others.

There has been significant progress in recent years in directed differentiation of pluripotent cells across a variety of tissues. What do you see as being the biggest obstacles in generating PSC-derived cells that are functionally equivalent to their primary cell counterparts?

To date, there has been a tremendous reliance on the mouse as a model of human development. Indeed, there is significant evolutionary conservation among gene regulatory networks, but we still have vanishingly little information about the transcription networks truly native to the developing human embryo. Only recently have studies begun to emerge, and such data has provided valuable insight, for example, into the sequence of gene activation in the developing human pancreas (see the work of Neil Hanley, Manchester). These data inform how directed differentiation protocols are tailored and optimized, and begin to help us understand the morphological and physiological differences between mouse and human islets.

Differentiation protocols have proven to be difficult to transfer from lab to lab. Do you see a role for protocol and reagent standardization enabling effective collaboration and the ability to make cohesive contributions to the field at large?

Standardizing reagents certainly help "level the playing field" when labs culture and differentiate hESC/hiPSC identically, but the other lingering issue is variability among hESC/hiPSC lines. We encounter, like many labs globally, variation in differentiation efficiencies of hESC/hiPSC, which inevitably requires tweaking and modifying differentiation platforms to accommodate persnickety hESC/hiPSC personalities.

Do you see hiPSCs having an impact on the treatment of diabetes in the future? What technical hurdles need to be overcome in order for hiPSC-based cellular therapies to become a reality?

I'm indeed very hopeful given the trio of papers published in the last year or so demonstrating improved differentiation of human pluripotent cells, both hESC and hiPSC, into "functional" beta cells (work from Melton, Kieffer and Hebrok). Of course, there are downstream hurdles to overcome, including proper encapsulation and immune tolerance.

On STEMCELL Technologies Products

What STEMCELL products do you use in your lab?

We use mTeSR1 media for culturing human Embryonic Stem Cells (hESC) and induced Pluripotent Stem Cells (iPSC), and the STEMdiff Pancreatic Progenitor kit for differentiating pluripotent cells into pancreatic progenitors.

Is there a product that you have found to be particularly useful in carrying out or optimizing your experiments? Can you describe your experience?

The STEMdiff kit is useful for generating PDX1+/NKX6.1+ pancreatic progenitors from different hESC/iPSC lines as the protocol is easy to follow and in our hands consistently yields 80% or more double positive cells.

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On David

Tell us about yourself:

I have a PhD from the University of St Andrews, and now I'm the Principal Investigator at the University of Edinburgh's MRC Centre for Regenerative Medicine. For the last decade, I've worked in the field of pluripotent stem cell biology and hepatocyte derivation.

My work is focused on human stem cell differentiation. We're interested in developing predictive human models and new medical devices. We have developed a robust stem cell differentiation process which generates high fidelity human hepatocytes. More recently, we have combined our approach with materials chemistry to deliver a more functional and stable in vitro system. This work has led to a University of Edinburgh spin-out company, FibromEd, which focuses on developing novel tools for research and industry.

On PSC Research

What do you consider to be the most important advance(s) in stem cell research over the past 5 years?

Given our focus on cell based modelling, I would say the most important advances in our field are:

• a. Generation of human iPSCs from somatic cells permits selectable genotype.
• b. Generation of efficient differentiation processes and disease models that recapitulate human physiology.
• c. Demonstration that materials chemistry has an important role to play in the maintenance and differentiation of stem cells.

What advances do you hope the field will achieve in the next 5 years? See More

I hope that in the next five years that we will gain a better understanding of human physiology using stem cell derived somatic cells as models. Moving on from this I can envisage the development of gold standard models that allow the identification and development of safer and more efficacious medicines.

How challenging was it for you to produce a robust and reproducible differentiation protocol to generate end-stage terminally differentiated hepatocytes from hPSCs?

We generated a number of differentiation procedures to get to where we are today. The earliest prototypes relied heavily on chemical additives to drive cell commitment. While this was efficient, the breakthrough came with the addition of key developmental factors to drive cell commitment. This not only improved the efficiency of our process, but cell function was substantially improved in vitro and in vivo (Hay et al, 2008, Proc Nat Acad Sciences, 105, 12301-12306). Additionally, these studies served as the blue print to efficiently deliver iPSC-derived hepatocytes from different genetic backgrounds (Sullivan et al, 2010, Hepatology, 51(1):329-35). A further breakthrough came using synthetic polymer surfaces to stabilize and improve stem cell-derived hepatocyte biology (Hay et al, 2011, Stem Cell Research, 6: 92-101) to the point where the cell based assays are now suitable for industrial screening.

Some recent studies have revealed important small molecules that can replace growth factors within expansion and differentiation of hPSCs. How do you foresee the effect of such small molecules, and what is your view of their long-term effect on culture systems and the cells themselves?

I think the use of small molecules which accurately mimic growth factor action has a very important role to play in stem cell scale up and differentiation. Such approaches have the potential to provide defined cell culture conditions, which will lead to improvements in biological modelling and deliver affordable research and clinical grade somatic cells. However, one must be careful and thoroughly examine any potential off-target effects and/or cellular stresses. Therefore, I think more detailed studies are required to assess the long-term effects of chemical addition on cell stability. We are working toward this goal at the University of Edinburgh and FibromEd Limited.

On mTeSR™1

What has your experience been in working with mTeSR™1?

We were aware that we needed to move away from murine embryonic fibroblast conditioned medium (MEF-CM) to a quality assured and serum free stem cell maintenance system. Moreover, we required a growth medium which consistently supported manufacture at larger scale.

So we started using mTeSR™1 four years ago for our routine experimentation and scale-up of our differentiation process. mTeSR™1 has enabled our research by providing more defined conditions and less batch-to-batch variation that was observed with MEF-CM. mTeSR™1 has also permitted rapid scale up in a serum-free environment, enabling larger experiments to be routinely carried out in the lab. We have used mTeSR™1 successfully and have been able to maintain hESC populations for over 40 passages with stable karyotype.

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