The Tissue Modulation Laboratory

Layered cross-section of adipocytes cultured under macromolecular crowding. These fluorescence microscopy images demonstrate that crowding results in a massive buildup of extracellular matrix scaffolding (red) between adipocytes differentiated from human mesenchymal stem cells. Lipid droplets are shown in green (BODIPY); nuclei in blue (DAPI).
Implanted blood-derived angiogenic cells (BDACs; shown as green circles) marching along capillaries (tubular structures). We can source BDACs from blood using macromolecular crowding technology. BDACs can be used to help heal hind limb ischemia in nude mice (Blocki et al., 2015) and are an exciting new avenue for diabetic wound care.

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Macromolecular Crowding & Stem Cell Platform

Macromolecular crowding (MMC) is a multi-platform technology. The extracellular environments of pluricellular organisms contain a high concentration of macromolecules: proteins, DNA, lipids, and polysaccharides. Growing cells on culture plastic in dilute aqueous media hardly compares to the macromolecularly crowded microenvironments from which they originate. To create conditions that come closer to tissue physiology, we have developed low-viscosity culture additives (large polyssugar macromolecules) that dramatically enhance extracellular matrix deposition. This enables both fully differentiated cells and stem cells to build their own complex microenvironments. The resulting dynamic cell-matrix reciprocity fuels profound beneficial effects on mesenchymal stem cell (MSC) cultures, including substantially increased proliferation rates while retaining differentiation capacity.

Once we had established the effectiveness of crowding in enhancing an astonishing variety of cell culture properties, we began to apply this novel technology across our research areas. We now use crowding to build adipose tissue from MSCs for metabolic studies, to generate mesenchymal progenitor cells from peripheral blood, and to aid the building of stable tissue constructs. We also continue to study the biophysical effects of macromolecular crowding in simpler models and by computer simulation.

Biophysics of Macromolecular Crowding

We study the underlying biophysical effects of macromolecular crowding in simpler models and computer simulations. We have modelled DNA hybridization under crowded conditions and have simulate the effects of mixed macromolecular crowding, i.e. usage of more than one size species of crowders (Dewavrin et al., 2015). We are studying the micro-architectural effects of collagen assembly under crowded conditions and exploit this to build crowding gradients to influence collagen assemblies (Dewavrin et al., 2014).

Stem cell technologies

We are deeply interested in studying and exploiting the effects of extracellular matrix deposited under crowded conditions on the proliferation and differentiation of human adult stem cells. Our publication series on stem and progenitor cells began in collaboration with MIT (Zeiger et al., 2012), where we showed the influence of matrix formation via MMC and its influence on cytoskeletal orientation, as well as the effects of matrix made under crowding on the propagation of human embryonic stem cells (Peng et al., 2012). We have demonstrated that, and how, MMC amplifies adipogenesis of MSCs via cell-matrix reciprocity, confirming Mina Bissell's postulate from the 80s (Ang et al., 2014) - see animation at the top of this page. We have shown with Lonza and patient cells from the National University Hospital Singapore that MMC drives stem cell proliferation. The effects can be related to a beneficial microenvironment formed under MMC and has enabled us to secure substantial funding and industry interest. In the framework of a 5 million dollar (SGD) Bench & Bedside grant, we are currently working on the generation of best-in-class MSCs using MMC, potentially in combination with small heparan sulfate sugars (collaboration with Simon Cool and Victor Nurcombe of IMB, A*STAR, and James Hui of the NUHS Department of Orthopaedic Surgery).

Platform to differentiate pericytes from peripheral blood

We have also shown that pulsed MMC allows the generation of a pericyte-like angiogenic phenotype derived from monocytes from peripheral blood (Blocki et al., 2015). We have termed cells exhibiting this phenotype "blood-derived angiogenic cells" (BDAC). This work has strong therapeutic potential and is currently being evaluated in a hind limb ischemia model to make way for a larger project on critical ischemia treatment in human patients. Current preclinical work, also employing MRI for small animals, shows an acceleration of revascularization of ligated limbs but also a protective effect on skeletal musculature, which suggests additional effects of BDAC on myocytes or satellite cells (Blocki et al., 2015). We are currently evaluating the effect of injected BDACs on muscle degeneration and ischemia protection (see video above). Our dream would be to treat stroke patients with autologous pericyte-like cells. This is an ongoing collaboration with Prof Kishore Bhakoo of the Singapore Bioimaging Consortium, A*STAR.

TML research on BDACs featured in The Straits Times, 20th August 2015.

How does crowding work?

No single macromolecular species occurs at a high concentration, but taken together, macromolecules occupy a significant fraction (10 to 40%) of the total volume in vivo (up to 400 g/L).

The volume occupied by macromolecules is not accessible to other molecules. In addition, the volume between two neighboring macromolecules is partially unavailable to a similarly-sized molecule. Beyond a specific concentration of macromolecules, unavailable and occupied volumes add up to generate the Excluded Volume Effect (EVE).

EVE increases and redistributes the total free energy of the solution, enhancing the yield of all reactions driven by a variation of entropy. See picture below for a graphical explanation.

The cell interior is crowded with various macromolecules (coloured circles). These macromolecules both occupy space themselves and mechanically block access to much of the remaining empty space, thereby further excluding some of the unoccupied volume from becoming occupied.

Artificial polymers such as Ficoll, dextran sulfate and PVP can be used as macromolecular crowders in vitro. Their effect depends on their size, shape and charge of the molecule used; they have been selected to be as inert and stable as possible.

In order to generate EVE, a minimum threshold concentration of artificial crowders is needed. At TML, we have perfected the use of Ficoll in assisting cell culture, and our work has shown that the ideal Ficoll additive contains both Ficoll 70 (at 37.5mg/ml) and Ficoll 400 (at 25mg/ml).

Typical reactions enhanced by macromolecular crowding are protein folding and aggregation, protein-protein interaction, polymerization, enzymatic reactions or hybridization. For example, real-time PCR amplification is greatly enhanced by crowding:

Macromolecular crowding significantly increases the amplification of mRNA achieved via RT-PCR.

Over more than ten years of research with macromolecular crowding, TML has come to appreciate the power of crowding both in providing more physiological cell culture environments and in simply increasing experimental yield. The breadth of the applicability of macromolecular crowding is truly remarkable. Don't ask what crowding can do for you - rather, ask what it can't do!