You can have an individual neuron that has a hundred thousand connections to a hundred

thousand different cells.

The signals that come in to that cell are amazingly complex.

If you ever hope to understand disease, ever hope to understand how drugs function you

need to understand how they function; where all those connections are intact.

When you see a traditional anatomy display like this, you just see how far

science has come.

Today, we can even sequence single cells.

This is a frontier that the pioneers of medicine couldn't even imagine.

That's why we're here in Philadelphia, to meet Jim Eberwine, who is a pioneer in single

cell sequencing.

He'll show us how it's done.

What is the primary question you're now trying to address?

t revolves around understanding how different cells function within their natural micro-environment.

Each cell is different from each other cell.

If we can assess quantitatively these different biological features within these individual

cells.

That's one way of building up to the system and understanding it.

How does the method work?

Fortunately, here at Penn, we have a great collaboration with our

neurosurgery department.

We generally get human cortical samples for many of the neurosurgical specimens.

We also are able to get the hippocampal area.

It's in the hippocampus, which is the site of learning and memory in most mammalian systems-

there are millions of cells within this area, and we go to individual cells that are connected

to different neurons to see what the transcriptome or the genome looks like in those cells as

a function of the different types of diseases.

The key is being able to isolate the material from that individual cell.

For disperse cells in culture, using the patch pipette like a needle is very useful.

The problem with primary cell culture is you remove the cells from all the neighboring

cells, so those hundreds of thousands of different connections

are lost.

We developed a procedure called TIVA, [TIVA compound] which is transcriptome in vivo analysis,

that allows us to isolate the RNA from any individual cell [Live cell] in its natural

micro-environment [Cell loading] we can put our TIVA compound in to the cell, it will

go in through a chemical reaction, and then we focus light on a cell that's of interest

to us [Uncaging] the chemical compound breaks apart.

We can see that with the change in the color of the cell, and once we see that change,

we know that chemical can then [Annealing] bind to the RNA in that individual cell.

We can then take that entire tissue piece, grind it up, and the only RNA that we're going

to isolate [Capture] is RNA from the cell where this TIVA

compound has been activated.

[mRNA]

We identified the cell of interest, and then we activate TIVA only in this cell.

We shine the laser now, and then activate.

After this activation we can isolate this and then we will have full information of

their transcriptomics.

Even though in the same culture system, they showed the different physiological response,

[1:21:03:01] but once we get the transcriptomics information, we can understand why they are

different.

once we identify specific gene sets [1] or specific genes, we can actually [2] introduce

those transcriptome itself, instead of modifying the entire genome.

We came up with phototransfections which is using [3] high-intense laser light to making

transient pores into the cell.

Once we introduce the small hole, [4] you will see in the middle it will increase because

these green dyes are getting to the cell, Immediately transiently change from this cell

type to this cell type, so based on this information and analyses we can actually explain ... what

kind of information, imaging results are showing.

Now we have chance to define those, based on the single cell study, combining with the

imaging technology, and the RNA sequencing technology, and bioinformatics, and they all

three work together to identify the same goal.

the biological questions that are coming up is, how many different cell types are there?

Are those natural cell types?

What processes drive those cell types, and how do we define those cell

types?

It's more like a community or ecology of those cells interacting together to produce the

system level, tissue level, organ level function.

What we're really doing now is studying the ecology and systematics of the individual.

What inspires you about this work?

Each cell is different from every other cell.

It's the puzzle.

You have all this data, you know there are answers within that data.

The data itself is not knowledge.

You have to sieve through that data to try to gain that

knowledge.

It is our fondest hope, our greatest hope, that we'll be able to gain insight

into these diseases from that data.

It's incredible to think that we can actually manipulate single neurons.

Even amongst seemingly identical cells, there is this functional diversity that may explain

the operation of tissues and how they respond to disease.

Now we finally have the tools to unravel the collective behavior of cells.