March 2020: Meet the De Nooij Lab

Background:

What is the main focus of your lab?

Our main focus is on the development of the proprioceptive sensory system, which is the sensory system that informs the brain where your body and limbs are in relation to one and other. Our primary model system is the mouse, but about a year before I started my lab we also began modeling sensory neuron development, and diseases that affect sensory neurons (including proprioceptors), using stem cell based approaches.

How long have you had your lab? When did you join Columbia University?

I joined Columbia about 200 BC, but I started my lab in the summer of 2017, so that is somewhere between two and a half and three years.

How big is your lab currently?

At the moment we are five people in total. Eti, is a postdoctoral fellow who is modeling Friedreich Ataxia using FA-patient derived mechanoreceptors and proprioceptors (specifically trying to understand why these two sensory neuron subclasses are so much more vulnerable to the disease compared to nociceptive pain sensory neurons). Shirley is a research associate (and a master student in public health) who is working on improving our differentiation protocols (increasing yields and obtaining more mature neurons). Natalie is a current rotation student from the NB&B program who is exploring protocols for the hESC/iPSC derivation of peripheral glia such as DRG satellite cells or Schwann cells (to test to what extent FA disease may have a non-cell autonomous component). I myself am taking care of the in vivo mouse studies (understanding the molecular basis of the different proprioceptor subclasses). For this last project we are currently doing a lot of single cell RNA sequencing and Gautam, an undergraduate volunteer in the lab, is helping us with some of the bioinformatics analysis.

Where is your lab located?

We just moved from the CU Motor Neuron Center in VP&S to the 3rd floor of the William Black building. We are not yet fully set up, but we are already very pleased with the additional space in the lab and especially our own TC room!

Current affairs:

What are the most exciting projects/directions in the lab at this moment?

There are two aspects of the lab/our work that I am very happy about at the moment. With respect to our mouse project, we have long struggled to obtain genetic access to individual types of proprioceptive neurons. In the last couple of years however, we were able to develop some new genetic tools that, together with the technical advancement of single cell RNA sequencing, have enabled us to identify the relevant molecular markers. This means we finally can move on and start asking (and hopefully answering!) questions about the circuitry and function of distinct proprioceptor subclasses.

The other aspect I am very excited about is that our stem cell work is ‘growing up’.  For a mouse person, the work with tissue culture cells always seemed so easy - you can generate more cells in a short period of time, and freeze them if you do not need them for a while. Of course when you start working with cell cultures, or at least with ES/iPS cells, you realize they can present just as many challenges as working with mice. In fact, I now think they can be harder than working with mice; I mean, we are spending a fortune on all kinds of cytokines and special media to keep these cells happy and they still suddenly look bad for no apparent reason. Yet, while we remain to suffer some setbacks from time to time, I think that we are now getting in a place where we can start asking some more interesting questions with the system that we are building - and that is very exciting to me.

What are the biggest accomplishments that your lab recently had?

Right now it is securing the funding that we could move to the new lab space.

Technology:

What are the model systems that your lab is using? 

With respect to our mouse project we have various Cre/FlpO–dependent genetic reporters (mostly fluorescent) and various sensory neuron specific Cre or FlpO drivers.  For our stem cell work we are working with human ES or iPS cells, in which we have introduced fluorescent reporters that enable us to visualize nociceptors (TrkA-tdTomato), mechanoreceptors (MafA:tdTomato), or proprioceptive (Runx3:tdTomato) sensory neurons. Not all of these reagents have been published but we are happy to share with the right agreements in place.

What are the key techniques that your lab is using? 

When it comes to our stem cell work, I think for a large part we are still on the receiving end in terms of training, but we have developed protocols for the generation of different types of sensory neurons as well as some of the tools to validate the neurons.

We also ‘routinely’ perform single cell RNA sequencing (10x and plate sequencing) and use RNA scope (mostly on mouse tissue) to validate some of these data.

What facilities or equipment does your absolutely lab rely upon? 

In the lab we do not use a whole lot of equipment that is not standard for most labs. For experiments for which we need additional equipment (or expertise), we collaborate with other labs. With regards to core facilities, we use the JP Sulzberger genome center for all our single cell RNA sequencing projects and we also rely on both CSCI cores a lot. Barbara and the rest of her team at the stem cell core have been incredibly generous with their time and advice in guiding us through our various stem cell pluripotency scares. We also do all our FACS sorting with the CSCI flow core, with Mike and Daniel. (Mike keeps trying to get us to do his Flow course so we can become independent, but it is way too nice to sort with Mike).

Who shall be contacted with questions about equipment, resources and training?

Best to reach out to me directly: sd382@cumc.columbia.edu. 

Training:

What's your best approach to mentoring trainees in the lab?

I don’t think there is any one ‘best approach’ because what works for one trainee may not be appropriate for another trainee. I think my general goal is to try to create an environment where people feel comfortable to ask questions without having to be worried about whether it is a good or stupid question, and where people maintain a healthy perspective as to the relative level of importance of our work. (I mean look around you, there are so many things that we ought to be much more upset about than our failed experiment in lab). At the same time, I do demand a fair amount of responsibility from each lab member with regards to their own project. (For the record, even though I have come to adopt this CSCI term, I kind of dislike calling students and certainly postdocs ‘trainees’ - it feels a bit too belittling to me. In my experience, many students and postdocs at CU are beyond that stage and deserve to be taken more seriously).

Who were your most influential mentors/role models in science and what did you learn from them?

There are three people that I consider ‘role models’ in one way or another: My PhD advisor at the Massachusetts General Hospital (MGH) Cancer Center, Iswar Hariharan, the PI of our neighboring lab at MGH, Ed Harlow, and my postdoc advisor Tom Jessell. Iswar really emphasized the importance of reading papers, including outside of my field of study. Ed Harlow was amazing in exposing the most interesting aspect of a particular study (you’d be bored in a seminar until he asked a question that made you see how much potential the work had). Tom, was unparalleled in his attention to detail and ability to communicate the work of the lab in lectures or papers. I am never going to reach the level of any of these three mentors but they continue to inspire me to try to get as close as I possibly can.

What would be your career advice for students/postdocs?

Choose to work on project that truly fascinates you and be open to collaborate. Science can be too tough or lonely if you are not able to see the challenges as ‘fun’ at least 70% of the time.

Are you accepting rotating students at the moment?

We at capacity for our human disease-related stem cell work right now, but I’d consider accepting a rotation student who’d want to participate in the developmental  proprioceptor studies (using stem cells or mouse models).

Lab management:

Does your lab have any fun traditions?

My current lab members have been in the lab for less than a year so we have not yet developed much in the way of traditions.

What is the key to running a successful lab

I have no idea - check back in a few years!

What was the most exciting part about starting your new lab?

For me it was the opportunity to stay in academia and to make my own strategic decisions in pursuing my research projects.

Stem Cell Directions:

What are the most important recent developments in the stem cell field?

I may be too new to the field to judge if this development is in fact the most important but at the moment I am personally excited about the relevance of metabolic pathways during development. In my ignorance I have generally considered metabolic genes and pathways as part of the (non-interesting) ‘housekeeping genes’ category, but we now start to see that they can influence the epigenetic landscape, and control cell type lineage decisions. It is exciting that the use of human ES/iPS cells allows us to uncover and study these effects in much greater detail. In addition, I remain fascinated by our ability to generate ever increasingly complex tissues/organoids from stem cells.

Which stem cell conferences does your lab attend?

NY stem cell foundation, Keystone meetings on somatosenstaion, International Ataxia Research Conference

CSCI:

What was the main reason of you joining CSCI? What are the beneficial aspects of CSCI membership for your lab?

As a faculty member in a predominantly clinical department (Neurology), joining CSCI provided a great opportunity to interact more with other faculty performing basic research. In addition, being new to the stem cell field, it was nice to get to know other stem cell faculty, learn about their work, and to be able to tap into this network for advise on our own studies.

What do you plan to bring to the CSCI community?

A community only functions as a community if there are participating members so I do my best to attend as many CSCI seminars/WIP talks/activities as possible (although I have to admit this can be a bit of a challenge at times). Other than that, I hope that we can contribute a new model system to the existing expertise/models already available among the CSCI members.