Turning Stem Cells into Drug Factories


Wouldn’t it be nice to have cells that express the right molecules at the right place and the right time to augment or even initiate healing?

Researchers at the Brigham and Women’s Hospital and Harvard Stem Cell Institute have inserted modified messenger RNA to induce mesenchymal stem cells to produce adhesive proteins  (PSGL-1)and secrete interleukin-10, a molecule that suppresses inflammation. When injected into the bloodstream of mice, these modified stem cells home to the right location, stick to that site, and secrete interleukin-10 (IL-10) to suppress inflammation.

Improving MSC therapeutic potential viamRNA transfection with homing ligands and immunomodulatory factors. Illustration of (A) mRNA-engineered MSCs that express a combination of homing ligands (PSGL-1 and SLeX) and an immunomodulatory factor (IL-10), and (B) targeting mRNA-engineered MSCs to site of inflammation.
Improving MSC therapeutic potential viamRNA transfection with homing ligands and immunomodulatory factors. Illustration of (A) mRNA-engineered MSCs that express a combination of homing ligands (PSGL-1 and SLeX) and an immunomodulatory factor (IL-10), and (B) targeting mRNA-engineered MSCs to site of inflammation.

Jeffrey Karp, Harvard Stem Cell Institute principal faculty member and leader of this research, said this about this work: “If you think of a cell as a drug factory, what we’re doing is targeting cell-based, drug factories to damaged tissues, where the cells can produce drugs at high enough levels to have a therapeutic effect.”

Karp’s paper reports a proof-of-principle study has piqued the interest of several biotechnology companies, since it has the potential to target biological drug to disease sites. While ranked as the top sellers in the drug industry, biological drugs are still challenging to use. Karp’s approach might improve the clinical applications of biological drugs and improve the somewhat mixed results of clinical trials with mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have emerged as one of the favorite sources for stem cell therapies. The attractiveness of MSCs largely lies with their availability, since they are found in bone marrow, fat, liver, muscle, and many other places. Secondly, MSCs can be grown in culture for a limited period of time without a great deal of difficulty. Third, MSCs tend to be ignored by the immune system when injected. For these reasons, MSCs have been used in many clinical trials, and they appear to be quite safe to use.

To genetically modify MSCs, Karp and his co-workers made chemically modified messenger RNAs (mRNAs) whose bases differed slightly from natural mRNAs. These chemical modifications did not affect the recognition of the mRNA by the protein synthesis machinery of the MSCs, but did affect the recognition of these mRNAs by those enzymes that degrade mRNAs. Therefore, these synthetic mRNAs are very long-lived and the transfected cells end up making the proteins encoded by these mRNAs for a very long time. RNA transfection does not modify the genome of the host cells, and this makes it a very safe procedure, since the engineered cells will express the desired protein for some time, but not indefinitely.

The mRNAs introduced into the cultured MSCs included mRNAs that encode the IL-10 protein, which is cytokine that suppresses inflammation, the PSGL-1 protein, a cell-surface protein that sticks to the P-and E-selectin receptors, and the Fut7 gene product.  FUT7 encodes an enzyme called fucosyltransferase 7, which adds a sugar called “fucose” to the PSGL-1 protein and without this sugar, PSGL-1 cannot bind to the selectins.  Selectins are stored by cells and during inflammation, they are sent to the cell surface where they can bind cells and keep them there to mediate inflammation.  By expressing PSGL-1 in the MSCs, Karp and his group hoped to that the engineered MSCs would bind to the surfaces of blood vessels and not be washed out.

e-selectin_binding

Oren Levy, lead author of this paper, said, “This opens the door to thinking of messenger RNA transfection of cell populations as next generation therapeutics in the clinic, as they get around some of the delivery challenges that have been encountered with biological agents.”

A problem that constantly troubles clinical trials that use MSCs is that they are rapidly cleared from the bloodstream within a few hours or days after they are introduced. The Harvard team showed that rapid targeting of MSCs to inflamed tissue produced a therapeutic effect despite rapid clearance of the MSCs.

Karp and his colleagues would like to extend the lifespan of these cells in the bloodstream and they are presently experimenting with new synthetic mRNAs that encode pro-survival factors.

“We’ve interested to explore the platform nature of this approach and see what potential limitations it may have or how far we can actually push it. Potentially we can simultaneously deliver proteins that have synergistic therapeutic impacts,” said Weian Zhao, another author of this paper.

Protecting Blood Cell-Making Stem Cells from Cancer Drugs and Radiation


Our bone marrow serves as the nursery for blood cells. All the circulating blood cells, red cells, white cells and platelets, are derived from a rare population of bone marrow cells known as hematopoietic stem cells or HSCs. Normally, the majority of HSCs rest and take a break while a small fraction of them proliferates and differentiates into progenitor cells that differentiate into mature red and white blood cells.

When the body needs more blood cells, for example after blood loss or during inflammation, more HSCs proliferate. However, what tells a HSC to cool it and take a powder or wake up get up and get dividing remains mysterious. Nevertheless, we do know one thing: location, location, location. Within the bone marrow are specialized site called “niches” that direct the behavior of the HSC. Quiescent niches, for example, house resting HSCs but other niches, known as vascular niches, are located in close proximity to the blood vessels that line the bone marrow sinusoids and harbor the minority of hard-working HSCs.

A new paper in Nature Medicine by Ingrid Winkler from the Mater Medical Research Institute and various other collaborators has clarified one of the mechanisms that tell HSCs to keep proliferating. A cell adhesion molecule called E-selectin is expressed by some bone marrow blood vessel cells (endothelial cells) in those blood vessels close to that thin layer of connective tissue that lines that the medullary cavity (endosteum).

E-selectin is normally expressed in endothelial cells that are experiencing inflammation. Inflammation is the result of tissue damage, and inflammation induces the synthesis E-selectin in endothelial cells. When white blood cells sense that damage has occurred that they are needed to fight off invading microorganisms, they bind to inflamed endothelial cells and crawl between them. Selectins, in general bind to sugar residues on the surfaces of cells, and are, therefore, members of a larger group of sugar-binding proteins known as lectins.

Back to the bone marrow: When mice that genetically lacked E-selectin were examined, they had fewer proliferating HSCs in their bone marrow. Was this due to problems with their HSCs? Clearly not, because when the HSCs from the E-selectin-deficient mice were transferred into normal mice, they divided perfectly well, and when normal HSCs were dropped into the bone marrow the E-selectin-deficient mice, they also failed to proliferate very much. The difference in these mice is their bone marrow microenvironments and not their stem cells.

HSCs divided less in E-selectin-deficient mice, but when normal mice were treated mice small molecules that inhibited E-selectin aged slower and also were protected against toxic stress-inducing conditions such as radiation and chemotherapeutic drugs. Inhibition of E-selectin inhibition improved mouse survival and recovery after radiation and chemotherapy.

As you might guess, E-selectin inhibitors are already in clinical trials for sickle-cell disease. This might very well accelerate the translation of these data such as these to the clinic. Just think, treating patients with E-selectin inhibitors before radiation or chemotherapy might protect the quiescent HSCs from the toxic effects of cancer therapeutics. Also, treatment that induces HSC quiescence also reduces leukocyte production, and could also elicit anti-inflammatory effects by reducing the systemic supply of leukocytes. These data from Winkler’s paper might be the impetus for designing ways to protect patients from the side effects of cancer therapy.

See I. G. Winkler et al., Vascular niche E-selectin regulates hematopoietic stem cell dormancy, self renewal and chemoresistance. Nat. Med., published online 21 October 2012 (10.1038/nm.2969).