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Melika Sarem, PhD

University of Freiburg, Germany

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Biography

Dr Melika Sarem is a Project Leader/Postdoc in the area of Biomedical Sciences and Regenerative Technologies in the Institute for Macromolecular Chemistry at the University of Freiburg, Germany. Melika received her master's degree with honors in Biomedical Engineering, from Amirkabir University of Technology, Tehran, Iran. She then earned her PhD (summa cum laude) in Natural Sciences from University of Freiburg in 2017 under supervision of Prof V Prasad Shastri, specializing in biomimetic materials and stem cell biology. Her doctoral thesis "Role of Intrinsically Disordered Phosphoprotein Secondary Structure in Bone Biomineralization and Impact of Biomimetic Apatite on Endochondral Ossification" received The Best Doctoral Dissertation Award (Arthur-Lüttringhaus-Preis-2018) from University of Freiburg. In her doctoral effort Melika developed biomimetic system to engineer bone-like hydroxyapatite, and using this system she investigated how bone mineral phase impacts human bone marrow derived mesenchymal stem cells (MSCs) fate. She discovered bone mineral phase stimulates extracellular calcium sensing receptor (CaSR) in MSCs, and the hyperstimulation of this receptor blocks endochondral ossification and strictly promotes formation of bone via intramembranous ossification in MSCs.

Currently, Melika works on several multidisciplinary projects at the intersection of biophysics, materials science and developmental biology with a translational focus that incorporates various cutting edge technologies such as 3D bioprinting.

Melika's research has resulted in several publications in highly prestigious journals including Advanced Materials, Proceedings of the National Academy of Sciences - USA (PNAS) and Small, which has been extensively covered by the press. Her research impact in orthopedic tissue engineering and regenerative medicine has also been recently recognized by orthoregeneration network (ON) via ON/EORS education scholarship.

Learn about Melika’s research

Title: Doing more with less: Stem cells regulate their fate by altering their stiffness

Learning Objectives

  • Role of biophysics in mesenchymal stem cell biology
  • Mechanosensing proteins in chondrogenic differentiation

Although mesenchymal stem/stromal cells (MSCs) chondrogenic differentiation has been thoroughly investigated, the rudiments for enhancing chondrogenesis have remained largely dependent on external cues. Since aggregation of MSCs, a prerequisite for chondrogenesis, generates tension within the cell agglomerate, we theorized that the initial number of the cells within the aggregate could function as an intrinsic activator of a mechanobiology paradigm and alter the outcomes.

We discovered that reducing aggregate cell number (ACN) from 500k to 70k leads to activation and acceleration of the chondrogenic differentiation, independent of soluble chondro-inductive factors, via β-catenin dependent TCF/LEF transcriptional activity and expression of anti-apoptotic protein survivin. Our state-of-the-art mechanical testing revealed a correlation between progression of chondrogenesis and emergence of stiffer cell phenotype. In-depth Affymetrix gene array analysis proposed that the down-regulation of genes associated with lipid synthesis and regulation could account for observed outcomes. Furthermore, we illustrate that implanting aggregates within collagenous matrix not only decreases the necessity for high quantity of cells but also leads to drastic improvement in quality of the deposited tissue.

In summary, our study presents a simple and donor-independent strategy to enhance the efficiency of MSCs chondrogenic differentiation and demonstrates a correlation between MSCs chondrogenesis and mechanical properties with potential translational applications.

Watch the webinar

(00:00)

Slide 1

Moderator:  Hello everyone, and welcome to today’s live broadcast, Doing More with Less: Stem Cells Regulate Their Fate by Altering Their Stiffness, presented by Dr. Melika Sarem, Project Leader/Post-Doctoral Associate, Biomedical Sciences and Regenerative Technologies, Institute for Macromolecular Chemistry, University of Freiburg, Germany.  I’m Christy Jewell of LabRoots and I’ll be your moderator for today’s event. (00:26) Today’s educational web seminar is brought to you by LabRoots and sponsored by Thermo Fisher Scientific.  To learn more about our sponsor, please visit thermofisher.com/cellcultureheroes.  Before we begin, I would like to remind everyone that this event is interactive.  We encourage you to participate by submitting as many questions as you want, at any time you want during the presentation.  To do so, simply type them into the Ask a Question box and click Send.  We’ll answer as many questions as we have time for at the end of the presentation. (00:57) If you have trouble seeing or hearing the presentation, click on the Support tab found at the top right of the presentation window or report your problem by using the Ask a Question box.  This presentation is educational and thus offers continuing education credits.  Please click on the Continuing Education Credits tab located at the top right of the presentation window and follow the process to obtain your credits.  And now I’d like to introduce our presenter, Dr. Melika Sarem. (01:26) For a complete biography on Dr. Sarem, please visit the Biography tab at the top of your screen.  Dr. Sarem, you may now begin your presentation.

(01:40)

Dr. Melika Sarem:  Hello everybody, thank you so much for joining our webinar today.  It’s a great pleasure to have you all here.  My name is Melika Sarem, and I’m a Project Leader at the University of Freiburg at Institute of Molecular Chemistry and BIOSS Centre for Biological Signaling Studies.  Today I will talk about one of my recent publications, which was published three months ago, in Stem Cell Research and Therapy journal.  My publication was highlighted in several news agencies, with an article entitled Doing More with Less: Stem Cells Regulate Their Fate by Altering their Stiffness. (02:11) So if you’re interested, let’s dive in.

(02:14)

Slide 2

One of the biggest motivations of my study is a disease called osteoarthritis.  I’m sure that most of you may know someone in your extended family and friends circle who suffers from osteoarthritis.  In the USA alone, 10 percent of men and 13 percent of woman, age 60 and over, have been diagnosed with knee osteoarthritis.  So what is osteoarthritis exactly?  It’s a degenerative joint disease, with is characterized by cartilage degeneration and osseous overgrowth. (02:52)  Cartilage, or better said, articular cartilage is a white (inaudible @ 0:02:57) tissue which covers end of bone and joint.  It enables bones of a joint to easily glide over one another, with very little friction.  Acting as a cushion between joints, cartilage can help distribute the load of pressure and weight over the surface of joint.  It also can serve as a shock absorber and it’s mainly composed of (inaudible @ 0:03:21) collagen.  As you know, unfortunately cartilage has a very limited self-repair and renewal  capacity, due to its avascular nature.

(03:28)

Slide 3

So what are the available solutions or treatments for osteoarthritis?  Of course one of the commonly prescribed medications are pain killers.  Pain killers could be a temporary solution for elderly people, but let’s not forget that osteoarthritis does not always happen due to wear and tear.  Many young adults are also diagnosed with osteoarthritis stemming from sport injuries, genes and overweight.  Currently, replacement of an articulating joint with a synthetic prosthesis represents the optimal treatment for end stage joint disease. (04:04)  However, it has its own limitation, as apparently even successful implants have failure rates of 20 percent after 10 to 20 years.  So as you see, it cannot work for really young adults.  There has a been a great interest in developing biological treatments for joint repair.  So what is next, what can be the next option?  One of these potential approaches is tissue engineering, which utilizes cells and biomaterials to regenerate tissue. (04:36)  Tissue engineering is divided to scaffold based and scaffold free methodologies.  In the scaffold based technique, cells are harvested from body (inaudible @ 0:04:49) in vitro and later (inaudible @ 0:04:48) scaffold and cultured in presence of bioactive molecules, and implanted in vivo.  However, scaffold free techniques recently, such as cell therapy and gene therapy, have attracted attention, more from scientific community. (05:04)  But you know what is very important here is that regardless of which approach is desirable for cartilage regeneration, one should know the development of process of cartilage, to be able to mimic the way nature does it, because as you know, nature does it really the best.  

(05:19)

Slide 4

So cartilage is made with a process called chondrogenesis.  I’ve divided this process to two stages.  The first stage starts with aggregation of mesenchymal stem cells, which starts with the formation of (stem @ 0:05:35) cell/cell contacts to adhesion proteins.  The initiation of this process (size bonded @ 0:05:41) and differentiation of closely packed mesenchymal stem cells is tied to regulated transmembrane adhesion proteins, such as in Caveolin.   Please remember that name, because you will hear it quite often in my talk.  The process leads to induction of chondrogenic phenotype and is regulated by several factors, such as growth factor hormone and mechanical forces.  This stage, cell/cell contact, plays an important role in transducing the mechanical forces into intra and extracellular biochemical cues to activation of signaling pathways.

(06:15)

Slide 5

So, what is our next step?  Stage two starts with progression of chondrogenesis and the position of matrix (inaudible @ 0:06:22) proteoglycans and collagen type two.  The process of maturation continues, and you can see in the cartoon which I have drawn, in this stage cells start to make distance from each other and reside in a space that they have created.  This space is called lacuna.  So you see cells are separated from each other, the matrix is reached with proteoglycans and collagen type two.

(06:44)

Slide 6

So how we can replicate this process in laboratory setting?  Thankfully it is very well established process.  Once you have accessed the mesenchymal stem cells, you can replicate this process in vitro easily.  One can obtain mesenchymal stem cells from different sources, such as bone marrow and fat.  But the ones we use in our study are from adult bone marrow.  They were kindly provided from Professor Eva Martin’s laboratory in (inaudible @ 0:07:15).  (07:16)  So what do after you isolate the cells?  First you have to expand them to have adequate cell number, then you detach them from flask and make mono dispersed solution.  And of course you have to count the cells.  Then you make solution of one million cell/ml in chondrogenic differentiation media.  In this chondrogenic differentiation media, you should include chondroinductive factors.  What are they?   They are called TGF beta, (inaudible @ 0:07:36) ascorbic acid, and etc. (07:41)  But the most and biggest promoter is TGF beta 1.  Then you add 500 µl of this solution in centrifuge tube, centrifuge at 800 rpm for 30 minutes and you are ready to go.  So that means every small centrifuge tube you have 500,000 cells.  Remember this number, because this is the gold standard number which so many scientists in different laboratories around the globe use it.  (08:08)  So you half-close the tube to make the oxygen and (inaudible @ 0:08:12) diffusion and keep in culture for 21 days.  After 21 days, you can cryo-section the pellets, visualize the proteoglycans in the matrix using histological staining, such as Alcian blue and sulphorino.  As you can see, the process is very easy and very well established.

(08:31)

Slide 7

So, in this picture you see the typical staining Alcian blue in the pellets.  There is one million pellets and another small satellite pellets.  Proteoglycans are staining blue and (cells @ 0:08:44) are stained using nuclear (inaudible @ 0:08:46) in pinkish-red.  So take a moment and look at this picture very, very carefully.  What attracts your attention in this very specific picture?  When I looked at this picture, I had a question why the satellite pellets close to the main pellets, shows stronger staining for proteoglycans, and the morphologically the chondrocytes are more mature.  (09:08)  You can see the formation of lacuna in the satellite small pellets.  This was a moment I would call it that the (inaudible @ 0:09:15) really hit my head.  I was thinking why this is happening, what is the reason behind it?

(09:20)

Slide 8

I had an interesting idea.  I thought maybe this is because of differences in the number of the cells within aggregated pellets, maybe this is more satellite pellets have less cell number. Because of that, it’s chondrogenically more advanced.  So I decided to make pellets with different cell numbers, starting from 700k to 500k.  I chose 700k as this size are the smallest ones that you can easily handle in the lab. (09:50)  And 500k, as I told you before, is a gold standard number that different scientists use.  As you see in this beautiful family picture, despite the fact that the pellets with the lowest initial cell number, which I (call it @ 0:10:04) from now, the initial cell number is the number of the cell which I put in this little centrifuge tube, which is here, for example, 70k, has seven times less cells comparison to the pellets with the highest initial cell number. (10:17)  After seven days, the volumetric size between the pellets is actually the difference is negligible.  After 21 days, the diameter of 500k pellets is only one-and-a-half times bigger than the 70k pellets.  So you see the production of matrix, which actually leads to increase of the size in the pellet, is more efficient in 70k pellets.

(10:41)

Slide 9

So the next step was to take a closer look in the pellets.  I want to if see the pellets (inaudible @ 0:10:49) almost similar size, they show different levels of chondrogenesis.  So I wanted to compare the expression of proteoglycans, which as I said the hallmark of chondrogenesis.  If you take a look at this Alcian blue staining, you will notice that generally from day 7 to day 21, in all the conditions, there is increasing intensity of the blue color.  (11:10)  And in both time points, with decreasing the initial cell number, pellets show a stronger expression proteoglycans and chondrocytes are looking more mature.  This is very interesting, the histological images demonstrate the impact of initial cell number on the organization and shape of the cells, and uniformity and intensity of the (inaudible @ 0:11:30) matrix is affected.  So you see here, when you change the initial cell number, not only the production is changing, but also the uniformity and intensity of the glycosaminoglycans and proteoglycans.

(11:42)

Slide 10

So, the very first idea that we had was that maybe this phenomena is due to deficiency in the nutrition diffusion in the bigger pellet.  Also additionally, hypoxia plays a very important role in profession of chondrogenesis.  So we thought that maybe the optimal chondrogenesis occurs in the pellet where there’s a proper balance between hypoxia and nutrition diffusion, so you have enough number of the cells to actually create hypoxic environment.  (12:14)  But also there are not too much that you can diffuse nutritions very easily.

(12:19)

Slide 11

So, we decided to ask for help from modeling scientists.  I believe after so many years working in academia, I believe that big science happens when you have a lot of collaborators from this discipline, because they can bring ideas, technologies and all the stuff that you can use to make your science more meaningful.  So Dr. Simon Tanaka from ETH Zurich helped us in this portion of the project and he modeled our pellets and assessed the diffusion of TGF-beta, glucose and oxygen in our pellets. (12:50)  Interestingly, his modeling analysis shows that oxygen concentration at the center of the pellet is expected to be similar to the edge of the pellet, with less than ten percent differences observed in the case of the 500k pellets.  So what we saw was actually the (inaudible @ 0:13:06) diffusion is not changing so much, the difference in oxygen concentration predicted for different pellets do not constitute hypoxia, as even we have larger variations in human physiology. (13:21)  So likewise the drop in the concentration of TGF-beta was also predicted to be extremely negligible.  However, his modeling data reported that the concentration of glucose is slightly lower in the pellets with the higher cell number.  Nonetheless, the absence of any remarkable differences in the diffusion of oxygen and nutrition in the pellets with various initial cell numbers pointed us that maybe there are other mechanisms involved in the data, which we are able to see.

(13:50)

Slide 12

But you know, you have to always prove the modeling.  The next step we tried to prove the modeling data.  So we stained for hypoxia inducible factor, which is called Hlf1alpha, and its expression is upregulated in hypoxic condition.  As you see in immunohistochemistry images do not really show appreciable differences between different pellets.  So you can see that in the pellets starting from 70k to 500k, the expression of Hlf1 alpha is not really changing dramatically.

(14:20)

Slide 13

So, in this stage we know what is not responsible, but we needed to know what is actually responsible for what we see.  So we decided to use Affymetrix gene array analysis.  I know it’s expensive, I know it puts a lot of burden on the lab, but it’s really worth it, because you can really get a lot of information about your research.  We used this Affymetrix gene array analysis to get more insight into the difference of the gene expression level in different conditions. (14:50)  So I isolated cells from different pellets, 48 hour post initiation of (inaudible @ 0:14:54) differentiation, and as you can see in the map, this special heat map shows (inaudible @ 0:15:00) on the differentially regulated genes, as a function of changing the initial cell number within the pellets.  This Affymetrix gene array analysis showed us that the genes associated with chondrogenesis and endochondral ossification are up-regulated in pellets with lower initial cell number, which is actually very good news, (15:20) because we also saw that in the pellets after (inaudible @ 0:15:23) pellets after day seven and day 21, we showed that the pellets with the lower initial cell number actually show better chondrogenesis.  So the up-regulation of chondrogenic associated genes was quite expected.  But what is interesting is that (inaudible @ 0:15:38) of top 300 differentially regulated genes, we were able to find out that genes involved in regulation of lipid storage, such as Caveolin 1—remember the name again, this is very important protein, (15:50) interleukin 6, TG36, (inaudible @ 0:15:55) 3 and genes involved in regulation of lipid biosynthesis process and regulation of lipid transfer, such as LIP-10 and self-inducible kinase 1 were all down-regulated in pellets with lower initial cell number.  So we saw all these genes with 70k pellets.  Additionally, we also didn’t see any significant differences in expression of hypoxia associated genes, such as Hlf1 alpha 1 and 3. (16:20) between MSC’s from various initial cell numbers, which also confirmed our modeling data.  

(16:28)

Slide 14

So until now we had the hint and our hint was the lipid storage.  So let’s keep on working on that.  There is strong evidence that the mechanical properties of cells can contribute to fate choices.  The mechanical property of a cell is determined by different cellular components, such as plasma membrane and cytoskeleton.  It has been suggested that presence of cholesterol and polysaturated fatty acids in the lipid bio-layer increases the cell stiffness. (16:58)  We have shown in our own lab recently, that lipid cells that alter cell plasma membrane in bio-layer composition, and this impacts the deformability of the cell membrane.  Since several genes associated with lipid transfer and lipid storage were down-regulated in pellets with lower initial cell numbers, the 70k and 150k, we investigated a few differences at the gene expression level.  It translated into changes in the stiffness of the cell.  This is very, very exciting and (inaudible @ 0:17:25) we hit a jackpot. (17:30)  So we decided to ask for help from the Dr. Oliver Otto, who has recently developed a new technology to assess the mechanical properties of thousands of cells in less than a minute.  This fascinating technique is called Real-Time Deformability Cytometry.  It works based on hydrodynamic deformation of cells translocating to a microfluidic channel in a contact-free manner, and how cool is that?  RTDC is able to analyze more than 100 cells per second in real time. (18:00)  So to do this experiment, we almost have to actually travel across Germany.  We are living in (inaudible @ 0:18:07), which is in southwest of Germany and Dr. Oliver Otto is actually currently in Greisbach, which is northeast.  So we traveled across the country to analyze our sample.  But believe me, it’s definitely worth it.  

(18:20)

Slide 15

I think before going through the data, I often use an RTDC spreader to describe a little bit how we did the experiment.  So first I digest my pellets in keratinase to get the single cell suspension.  Then we analyze the cells using RTDC.  Our data shows that cells extracted from 70k pellets are bigger than the cells extracted from other pellets, which is actually we were able to show this data in our histological images. (18:50)  As I told you before, the cells are more mature.  As chondrocytes become more mature, their size increases.  Then also RTDC shows us that there is a direct relationship between decreasing the initial cell number in the pellets and increasing the cell size.  But more interestingly, the cells extracted from 70k pellets were less deformable than any other cells extracted from other conditions.

(19:16)

Slide 16

So, Dr. Oliver Otto also helped us to analyze the elastic modulus of the cell.  As you can see in the bar graph, by decreasing initial cell number in pellets, elastic modulus is increasing.  This trend is even more appreciable at day 7 of chondrogenic differentiation.  So you can see the trend, by decreasing cell number, of increasing elastic modulus of cells, even in day 2.  But the trend becomes more significant and more and more clear by day 7 of chondrogenesis.

(19:50)

Slide 17

This was very interesting data.  We were very encouraged by it.  So we decided to take a look at the expression of mechanosensing proteins, with a known role in chondrogenic differentiation.  So we saw that the (inaudible @ 0:20:04) mechanosensing, let’s look more towards the proteins.  In order to ascertain the mechanical underpinning to regulation chondrogenesis, because of this difference in initial cell number, we investigated that the expression proteins involved in cell/cell contact. (20:20)  One of the proteins known to inhibit cell/cell contact is Caveolin.  It’s a main scaffolding protein residing in cholesterol rich membrane (inaudible @ 0:20:31) domains, which has a documented role in mechanic (transduction @ 0:20:34) and it’s also implicated in transduction of mechanical versus extra cell/cell junction (inaudible @ 0:20:39) activated channels.  This was the same protein which I mentioned to you before that you should remember the name.  Caveolin have been implicated in the compartmentalization and regulation of many signaling events, (20:50) such as MSC renewal and differentiation to the adipogenic and osteogenic differentiation.  If you look at the (inaudible @ 0:20:57) data, you will see that expressing Caveolin-1, you variously correlated initial cell number within the pellet, and chondrogenic potential of the cell.  In contrast, in Caveolin expression show the completely opposite trend, the pellet with lower initial cell number already showing appreciable expression by day two, which after seven days was two to threefold higher, compared to high initial cell number conditions.  (21:22)  Implying that increasing initial cell number, using MSC aggregation has negative impact on Caveolin expression and stabilization.

(21:31)

Slide 18

So we were able also to show similar trends using immunofluorescence staining, and you can look at the cross section image of the pellets, the expression of Cav-1 in red color is strongly (inaudible @ 0:21:44) higher cell number and it’s localized in the area of the less chondrogenic potential, over expression of (inaudible @ 0:21:49) is a strong (inaudible @ 0:21:50) aggregate with lower cell number and its expression is (upset @ 0:21:53) in the area with less chondrogenic potential.  It is within the pellets with higher initial cell number, like 500k and 250k. (21:60)  The increasing inverse relationship between these two proteins actually which have a role in mechanic transduction, provide evidence for us and for others to investigate to opposing mechanobiology mechanism responsible for directing chondrogenesis in mesenchymal stem cells.

(22:20)

Slide 19

I believe curiosity is the reason behind all the big discoveries, and at this stage of project I was interested to know all the big signaling molecules are involved in this phenomenon.  Another important act in mesenchymal differentiation is beta catenin.  It’s a transcription co-activator of canonical Wnt pathway.  You know, canonical Wnt pathway’s role in mesenchymal biology development, the biology and formation of lung, bones and cartilage are quite well established. (22:52)  So I decided to look deeper in the role of beta catenin signaling.  To investigate this, I made aggregates with the mesenchymal stem cell transfected with commercially available research RNA, which is called 7-PGC.  I think the very first paper was published in (inaudible @ 0:23:08).  It’s commercially available, so you can buy this plasmid.  This plasmid is able to express beta catenin TCF (inaudible) and I tried to transfect my cells and analyze them by (inaudible @ 0:23:23) for beta catenin transcription activity in different time frame.  Transected MSC in access of nuclear beta catenin expressed only (inaudible) protein. (23:30) However, from beta catenin binding to TCF/LEF, green fluorescent protein expression is induced in the cells.  Surprisingly, cells in the pellets with lowest initial cell number showed a threefold increase in the (inaudible @ 0:23:47) positive cells on the second day of differentiation.  At day seven of differentiation, it was already increased by the fivefold.  This intriguing finding, which correlates well with catenin expression, (inaudible @ 0:24:00) (24:00) provides evidence for us that mesenchymal stem cells in the condensation phase, which means up to seven days, the site of plasmid domain of catenin interacts with beta catenin, so it makes stable catenin and Caveolin complexes, which then leads to transliteration of beta catenin inside the nuclear (eye @ 0:24:16) and it starts a signaling cascade.  The absence of such a difference also in day 21.  So as you look at the data at day 21, you will see that there is no differences in beta catenin expression, in beta catenin signaling and that means which is consistent with the maturation of chondrogenesis process at the end (24:34) because after 21 days, all the cells were almost mature.  The data obtained using genetically modified cells also needs to be confirmed using biotype cells.  So what I did, I used florescence staining and I stained for beta catenin in different stages of chondrogenesis and as you see in the 70k pellet, we have nuclear translocation of beta catenin to the nuclei, which is not actually absorbed into the pellet of 500k.

(25:02)

Slide 20

So, the big question comes now.  If this is all about cell/cell interaction, then this phenomenon should happen even in absence of chondro-inductive factors.  So if I say this is only cell/cell interaction, and this is autonomous, it should not depend on whatever you have inside you medium.

(25:21)

Slide 21

So what I decided to do to answer this question, I removed TGF-beta out of my equation.  So I removed TGF-beta from my chondrogenic differentiation medium.  This way (inaudible @ 0:25:33) I was able to see that even in absence of such a strong chondro-inductive factor, decreasing cell number increases MSC chondrogenic potential.  As you see, at day seven already, the 70k pellet shows signs of chondrogenic differentiation, (20:50) which by day 21, in the 70k pellet, you see stronger expression proteoglycans and also cell maturation.  You can see with the white arrow I have shown how the cells are reside in that corner in day 21 of 70k, and they are actually missing at 500k at day 21.  The cells are more condensed together, they don’t show any expression of proteoglycans in 500k, which is reversed with 70k.  So as I see this experiment showed to me this process is definitely autonomous.

(26:22)

Slide 22

So, the next question was how we can employ these findings in teaching (inaudible @ 0:26:29) genetic medicine, because as I told before, one of my main motivations was osteoarthritis.  In the bone and cartilage tissue in gene (inaudible @ 0:26:37), the biggest problem is acquiring sufficient number of the cells for in vitro culture, and in vivo implantation, because it’s costly, it’s time consuming and actually this step becomes a very limiting step for clinical translation. (26:52)  Because if you want to have a sufficient number of mesenchymal stem cells, that means you actually need to passage them massively inside the lab.  That means you put them through the culture for a long time.  And MSCs have shown that if they stain a culture for a long time, if you passage them (inaudible @ 0:27:12), they may lose their chondrogenic potential.  So in the expense of losing MSC chondrogenic potential, you can actually increase the expansion time, (27:22) which is not really efficient for tissue engineering and genetic medicine.  To add to this issue, we investigated if implanting few pellets with a lower cell number in the collagenous matrix, will actually to superior outcome, in comparison with traditional methodologies of dispersing cells when you (inaudible) through automatic.  So the normal, traditional cartilage tissue engineering or any tissue in engineering, you disperse single cell suspension in the scaffolding your biomaterials. (27:58)  But what I tried to do instead of doing that, I make my pellets before and then I implant this pellet later inside the cartilage in its matrix.  So after two days keeping the pellets in the culture, I implanted four of my 70k aggregates inside this collagenous matrix.  While in the control condition I had one million mesenchymal stem cells.  If you wonder why one million, because one million is kind of a gold standard number for this four millimeter diameter collagenous matrix, which are available, you can commercially buy them. (28:30)  After (inaudible @ 0:28:34) cell chondrogenic differentiation, what I saw that in the cells within the pellets were fully mature hypertrophic chondrocytes and were incorporated in the matrix.  They deposit (inaudible @ 0:28:46) the tissue, which is highly homogenous.  But you have to remember that the fact that the total cell number within this pellet’s condition is almost four times less than what is happening in traditional conditions, which is the control. (29:00)  However, they were able to produce very uniform matrix.  You can actually see that the presence of putting this 70k pellets actually inhibited the formation of hypoxia in necrotic zone, which occurs through the diminished oxygen and nutrition diffusion in such a large construct.  When you have a construct with four millimeter diameter, you should expect that you will have a nutrition diffusion problem and also oxygen diffusion problems.  Actually, this finding validates the premise of our study, and demonstrated the framework for potential translation and applications.

(29:42)

Slide 23

So, with this slide I would like to conclude what I have talked about now.  So shortly I would like to say induction of a stable chondrogenesis mesenchymal stem cells is crucial for cartilage tissue engineering, and bone regeneration using (inaudible @ 0:30:00) paradigm.  My significant effort has gone into optimizing culture conditions, serial expressions that in vitro for generating adequate cell number for manipulating (inaudible @ 0:30:09) expense of loss of chondrogenic potential. (30:10) In this study, we developed a donor independent solution to address donor variability and loss of chondrogenic phenotype.  We have demonstrated a direct relationship between the cell numbers during mesenchymal stem cell aggregation and chondrogenic differentiation.  Furthermore, you are able to show that enhanced chondrogenesis correlates with the emergence of a stiffer mesenchymal stem cell phenotype, which is accompanied by regulation of proteins involved in mechanotransduction, such as Cav-1 and N-cad.  (30:42)  So it says translation potential of our findings in the proof of concept study, we were able to demonstrate that the chondrogenesis which we were able to achieve with 70k pellets, is superior to the conventional approach that can be achieved.  We can achieve this by using fourfold less cell numbers.  So our analysis also showed that aggregate approach yields superior outcome by inhibiting the formation of necrotic zone and increasing efficiency of matrix deposition.  The result of our study provides compelling evidence for the role of cellular mechanics in chondrogenic differentiation of MSCs in 3D aggregate, with implication for understanding the mechanisms involved in skeletogenesis and MSC-based regenerative therapies.

(31:26)

Slide 24

So last but not the least, I would like to give a very, very big thank you.  So the least starts my professor and my advisor, Professor Prasad Shastri, for all the time supporting me and my ideas and also paying for my research.  Dr. Oliver Otto, for doing experiment using RTDC.  Dr. Simon Tanaka, for his support with the modeling analysis and all the members, current members and previous members of Shastri Lab, which they provided amazing environment to work and have fun. (32:00)  Also I would like to thank all the funding agencies, Helmholtz Virtual Institute on Multifunctional Biomaterials for Medicine, the Excellence Initiative of the German Federal State Governments, and the Swiss National Foundation Sinergia grant, who generously supported my research.  Last but not the least, I would like to thank Gibco Cell Culture team for providing me an opportunity to discuss my research in such a nice platform.  So thank you so much for listening.  So the talk is open for questions.

(32:35)

Moderator:  Thank you, Dr. Sarem, for your informative presentation.  We will now start the live Q&A portion of our webinar.  Now, if you haven’t started submitting your questions, please do so now. Just click on the Ask a Question box located on the far left of your screen, and click send.  We’ll answer as many of your questions as we have time for.  Okay, we’ve got some great questions coming in, let’s start at the top.  Dr. Sarem, how many donors did you use to generate the data?

(33:01)

Dr. Melika Sarem:  Yes, that’s a really nice question.  Actually we used three donors, two male and one female, with the age range between 25 to 50.  The data I presented now are from the male donor from age of 26, but you can also look at the other donors for example, the female 50 which I have in the (inaudible @ 0:33:22) information of the article online.  I hope that answers your question.

(33:32)

Moderator:  Yes it does, and if you have additional question regarding this answer, please feel free to submit them.  Now let’s go to our next question.  Could we use the same protocol with urine derived MS stem cells?

(33:45)

Dr. Melika Sarem:  That is a nice one.  Actually, urine derived stem cells are kind of a new research topic, I think in the last less than a decade or so they have been popular.  I have seen some articles discussing their chondrogenic potential, and I believe if they had a chondrogenic potential, then this protocol should work with them.  The same protocol we have used for a different mesenchymal stem cells and we got a similar outcome.  So I believe these cells should not be also different if they show proper chondrogenic potential.

(34:20)

Moderator:  Dr. Sarem, have you checked apoptosis weight?

(34:25)

Dr. Melika Sarem:  Actually, in the article we  have not (inaudible @ 0:34:26), they can look at the apoptosis but we have looked for Survivin, which is the apoptosis inhibitor protein.  So I didn’t present the data here because I thought it would maybe make it more complicated and time consuming.  But if you look at the article end line, you will see that we used immunofluorescence staining, we stain for Survivin.  We were able to show that actually expression of Survivin is up-regulated in the pellets with lower initial cell numbers. (34:55)  So places which we have high chondrogenic potential, they have also higher expression of the Survivin.  More interestingly, I would also say that we saw inverse relationship between Cav-1 expression and the Survivin.  So in there for example, the pellets, the higher initial cell number, we saw a decrease of Survivin expression and higher expression of Cav-1, which means that of course it could not directly look at apoptosis, but it shows that the expression of Survivin went higher, the rate of apoptosis should be lower. (35:29)  Also we looked for the proliferation rate and we also saw high proliferation rate in the pellets with lower initial cell number, like 70k and 150k.

(35:43)

Moderator:  Let’s go to our next question.  What do you see as the next step in understanding the mechanistic underpinnings?  And my next question, where do you see this being applied?

(35:51)

Dr. Melika Sarem:  Oh, I always like the future outlook question.  As I mentioned in the talk, we had looked for several (inaudible @ 0:36:04) proteins, for example, Cav-1 and (inaudible @ 0:36:08).  Role of Cav-1 in MSC chondrogenesis is really not known.  We were the very first one who discovered all of Caveolin in MSC chondrogenesis. (36:19)  We’re actually currently trying to understand the (inaudible @ 0:36:20) regulation of Cav-1 during MSC chondrogenesis and how knock-out or knock- down of Cav-1 could impact MSC chondrogenesis.  This is basically the direction we are currently trying to go.  As I said in the beginning, our main target is stem cell therapy, stem cell based regenerative medicine, and we are seeing the use of this application in bone tissue engineering and cartilage tissue engineering.  I hope this answers your question.

(36:55)

Moderator:  Thank you, Dr. Sarem.  Now we’ll go to our next question.  What is the advantage of using RTDC technique, as compared to other techniques?

(37:06)

Dr. Melika Sarem:  You know, if you know the RTDC is actually technique which is used for understanding the cell mechanical property.  The other technique which has been used for I would say more than a decade, is AFM, which is Atomic First Microscopy.  With Atomic First Microscopy, you have to spend hours and days to sit on the machine and we’re able maybe to measure ten cells.  So you will be able to measure ten cells after days, and you will have a very small population to analyze. (37:36)  If you have a heterogeneous population of mesenchymal stem cells, I wish you luck for analyzing with AFM.  But with RTDC you can actually measure thousands of cells.  So for example, if you look at the article or the data I presented now, we have looked for more than a thousand cells, which will be analyzed.  Then you load your sample, you analyze and the process take less than a minute.  So the time that you load your samples, by the time you finish the measurement, it may be, I don’t know, three to four minutes per thousand cells. (38:10)  Afterward, of course analysis takes some time, but it’s not as time consuming as AFM.  So I would say you will have a huge population to look after and also you will have better statistics, which will help you to understand the heterogeneity of the population.  I think actually the main publication from Dr. Oliver Otto’s group, which was published in (inaudible @ 0:38:36), if I’m not mistaken, that also clearly shows what advantage this machine has and how you can use this for understanding not only the population of MSCs, but also cells from blood.  That is very interesting.  I would recommend you to look at the paper, it is really fascinating technology.  And you can guy the machine, also, it’s available to purchase, I guess.

(38:59)

Moderator:  Thank you, Dr. Sarem.  Now have you ever done embryonic stem cell based (inaudible @ 0:39:06) analysis based on matrix properties?

(39:09)

Dr. Melika Sarem:  Actually no, we have not looked with the embryonic stem cells, but as I said for this very specific project, we used adult (inaudible @ 0:39:18) mesenchymal stem cells.

(39:23)

Moderator:  I have a few questions that are asking about your Instagram account.  Could you share your Instagram?

(39:28)

Dr. Melika Sarem:  You can look up my Instagram account, it’s called Lady in Science, I am very, very delighted to share my journey in this account.  I share some scientific stuff, some daily life of a female scientist and I would really be happy to see you there, it’s called Lady in Science.  Then you may be able to find me with a hash tag.

(39:53)

Moderator:  Yes, and you can also click on the biography tab at the top of your screen.  Dr. Sarem’s Instagram and her LinkedIn are listed there, that you can connect with her, as well.  Okay, let’s go to our next question.  Are there any changes to mechanosensitive ion channels, like piezo1 and piezo2, as they were implicated in chondrocyte mechanosensation?

(40:20)

Dr. Melika Sarem:  That is a really interesting question, because my supervisor, Professor Shastri, actually discovered the role of piezo1 in mechanotransduction in neurons.  So we actually, when I was doing my Ph. D. and afterward my post-doc, I also looked for the role of piezo1, specifically not piezo2, on the role of MSC chondrogenesis.  So to be honest, we were not able to see huge differences in the role of piezo1 in mesenchymally centered based chondrogenesis, actually in the experiment that I did.  Also in our Affymetrix gene array analysis, we were not able to (inaudible @ 0:41:02) piezo1 with a meaningful differences.  So yeah, I would say we did Affymetrix gene array at day two when we look at the expression systems, after seven days of chondrogenesis, in 14 days of chondrogenesis, but I believe that maybe an earlier time point, much earlier, could play some role.  But our analysis didn’t show any meaningful differences, actually from the time point that I discussed.

(41:31)

Moderator:  Thank you, Dr. Sarem.  Now we’re getting so many great questions coming in.  If we run out of time for your questions, just a reminder that Dr. Sarem will answer your questions via email, following the presentation.  We have time for a few more.  Dr. Sarem, what was the passage number that you used for the study?

(41:48)

Dr. Melika Sarem:  The passage number is actually (inaudible @ 0:41:54) MSC biology and MSC (inaudible @ 0:41:56) therapy.  Were very lucky to be able to use passage two.  So we passaged twice on mesenchymal stem cell (in vitro @ 0:42:02) and then we used them for our culture.  I would say recommend to use mesenchymal stem cells up to passage four, but after that I would not use it, because normally I use the differentiation potential after passage four.

(42:16)

Moderator:  Did you test for trilineage differentiation potential of MSCs?

(42:21)

Dr. Melika Sarem:  Yes, of course, when you use MSCs you have to test them for trilineage differentiation.  We tested them for (isogenic or osteogenic@ 0:42:28), chondrogenic and adipogenic differentiation.  

(42:34)

Moderator:  Dr. Sarem, do you think higher number of cells could have induced oxidative stress in the cells?

(42:43)

Dr. Melika Sarem:  Could be.  I remember when we had this paper on the (inaudible @ 0:42:50), we also got similar questions from one of the (inaudible @ 0:42:52), talking about oxidative stress.  But to be honest, we didn’t see any differences in the genes associated with oxidative (inaudible @ 0:43:07) in our Affymetrix gene array.  Also as I mentioned before, we didn’t see much of a difference in oxygen diffusion in our samples, but of course to be (inaudible @ 0:43:21) study, maybe other, more elaborate experiments can be done and it could be discussed more.  But we didn’t see anything in Affymetrix gene array we did after two days of chondrogenic differentiation.

(43:35)

Moderator:  Thank you, Dr. Sarem.  We have time for a few more.  Did you see differences in expression for fibronectin?

(43:41)

Dr. Melika Sarem:  That was a good question.  Actually we looked at fibronectin expression in the febrile state and also in (inaudible @ 0:43:49) state.  So I developed assay in our lab, like ELISA assay for fibronectin for basically soluble fibronectin.  The protocol on how to use ELISA is also clearly describing the (inaudible @ 0:44:04).  You can go and develop in your lab, it’s cost really less and it’s really easy to do.  So we actually looked at, as I said, both soluble and febrile fibronectin for the (inaudible) we used, (inaudible) and blood, and we were able to see actually in the pellets with a lower initial cell number, which is 70k and 150k, they have higher expression of fibronectin, which also could make sense because fibronectin is related to chondrogenesis, as all of you know, and also it’s underpin of beta catenin expression.  So the fibronectin expression was also confirming the data from the beta catenin up-regulation in pellets with a lower initial cell number.

(44:52)

Moderator:  Yes, great questions coming in!  We have time for maybe two more.  Did you see any differences in proliferation of MSC?

(45:02)

Dr. Melika Sarem:  Actually yes.  If you remember, I discussed in the modeling data that we saw differences in glucose diffusion in between lower initial cell number pellets and pellets with a higher initial cell number.  So we saw the pellets with the higher initial cell number, our modeling predicted to be half of what is in the media,  (45:32) which is around 2.2 ml (inaudible @ 0:45:35).  But yeah, in the lower initial cell number (inaudible) we didn’t see these differences.  But you know glucose is a small molecule, so the only factor that can alter its diffusion behavior is consumption rate.  Because of that, we started to examine the proliferation of (inaudible @ 0:45:52) mesenchymal stem cells, as I mentioned before.  So we looked at proliferation capacity of MSCs and we were able to actually see that MSCs within—I mean, they are differentiated already—in the lower initial cell number pellets were actually showing high proliferation rate in comparison to the cells from higher initial cell number. (46:11)  So when you look at the proliferation rate of the pellets from higher initial cell number, we actually saw the number of cells remained almost constant.  However, in pellets with a higher initial cell number, we saw their (inaudible @ 0:46:25) increase almost fourfold after 21 days, which is very interesting because we have also previously shown the high percent of cellular cartilage is basically a way to go for engineering cartilage in vivo.  (46:36)  And also the data, you can see clearly, we have presented clearly in the manuscript, so if you want to get more information about the proliferation rate or understanding which methodology we used to look at the proliferation rate of mesenchymal stem cells, I would recommend you to look at the article online.

(47:05)

Moderator:  Thank you, and we’ll end with this final question.  You mentioned that there’s a slight difference is glucose diffusion.  Could you please elaborate on that a bit?

(47:14)

Dr. Melika Sarem:  Yeah, this was actually what I mentioned a little bit before.  So as I said, glucose is quite a small molecule.  The difference in glucose diffusion can only be coming from higher consumption rate.  And higher consumption rate comes from proliferative status of mesenchymal stem cells or any cell type.  Generally, mesenchymal stem cells, when they go through chondrogenic behavior, chondrogenic differentiation pathway, they decrease their metabolic activity.  So they actually decrease their consumption rate. (47:50)  To understand why the consumption rate of MSCs was increasing, or to have higher glucose diffusion in a lower initial cell number pellet, we looked at, as I said, to the proliferation factor of MSCs and we were able to show that MSCs with the pellets with the lower initial cell number have higher proliferation rate.  So I would say this is a link between glucose diffusion and also MSC proliferation potential in lower initial cell number pellets.

(48:25)

Moderator:  Thank you again, Dr. Sarem.  Do you have any final comments for our audience?

(48:30)

Dr. Melika Sarem:  Yes, I would like to thank everybody for joining here for the talk.  I really, really enjoyed to discuss my research with you.  I learned a lot of interesting stuff from your questions.  I would really appreciate you if you have further questions, to contact me either via my email or my Instagram account or Twitter.  I would also like to thank again the Gibco Cell Culture team for giving me such an opportunity.

(49:00)

Moderator:  Thank you again.

(49:01)

Dr. Melika Sarem:  (inaudible @ 0:48:51) [over-talking]

(49:03)

Moderator:  Very good.  Thank you again.  Before we go, I’d like to thank our audience for joining us today and for their interesting questions.  Just a reminder, questions we did not have time for today and those submitted during the on-demand period, will be addressed by the speaker via the contact information you provided at the time of registration.  Again, I’d like to thank Dr. Melika Sarem for  her time today and for her valuable research.  We’d also like to thank LabRoots and our sponsor Thermo Fisher Scientific, for underwriting today’s education webcast.  This web cast can be viewed on demand, LabRoots will alert you via email when it’s available for replay.  We encourage you to share that email with your colleagues who may have missed today’s live event.  Until next time, bye-bye.

End Presentation (49:44)

Get to know Melika

What was your favorite day thus far?
The day I defended my PhD.

On your days off, what do you do?
Travel

Favorite phrase?
"I have no special talent. I am only passionately curious." –Albert Einstein

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