Prospectus for Research

William Gunn

Investigating the role of MSCs in repair of bone.

Introduction

The Adult Stem/Progenitor cells called MSCs were first identified by Friedenstein in 1976 ¹, and since then they have been found to have mitogenic, anti-apoptotic, and immunomodulatory effects in a variety of tissues. In 2003, Gregory et al.² found Dkk1, a soluble Wnt antagonist, is a major regulator of cell cycle entry in MSCs, and Tian et al.³ found a correlation between serum Dkk1 levels and the presence of osteolytic bone lesions in multiple myeloma(MM) patients. This strongly implicated MSCs in the pathology of MM, but also suggested MSCs may be at the root of many conditions where bone repair is defective. My research has investigated the role of MSCs in these conditions.

Summary of Earlier Work

In Vitro Study of MSC Osteogenesis

I initially began studying the effect of Dkk1 on MSCs because I was interested in the cell cycle regulatory processes of stem and progenitor cells. The signals that regulate the transitions between the stem cell state, wherein any given cell is mostly quiescent, but the population is capable of self-renewing for the life of the organism, and the progenitor state, wherein the cells are capable of rapidly dividing yet retain multipotentiality, seemed like they must hold the keys to advancement of regenerative medicine. Dkk1 looked particularly like one of these signals. My initial experiments showed that MSCs express LRP6, the receptor for WNT that is antagonized by Dkk1, and Kremen1, the co-receptor. I found that Dkk1 bound to MSCs by preparing radiolabeled Dkk1 in an in vitro transcription reaction, and localized the binding to the Cys2 by preparing deletion mutants of the full-length transcript, and using them to compete away the binding of the full-length. Based on this, we prepared peptides from the Cys 2 region, assayed the peptides, and selected certain peptides for in vivo study. We eventually found, through western blotting of various mouse excreta, that the animals were clearing virtually 100% of the peptides through their kidneys in under 4 hours. Meanwhile, I continued with the study of Dkk1 in vitro. I designed an in vitro assay for osteogenesis that allowed for rapid screening of small molecule modulators of WNT activity by measuring membrane-associated alkaline phosphatase(AP). Using this assay, I screened several small molecule modulators of the WNT pathway, an important mechanism in osteogenic differentiation, and also the PPARɣ pathway, important in adipogenic differentiation. The hypothesis was that activation of WNT signaling would promote osteogenesis, whereas activation of PPARɣ would push the cells towards adipogenesis at the expense of osteogenesis. Initial experiments showed that WNT activation or PPARɣ inhibition promoted osteogenesis, but did not much more than culturing the cells in osteogenic medium alone. Later, we found that inhibition of WNT with additional Dkk1 did inhibit osteogenesis, and, in that context, restoring WNT signaling did restore normal osteogenesis. This suggested WNT signaling may be behind at least some deficient bone repair phenotypes.

MSCs and Cancer

As I was developing the assay for measuring the effects of WNT modulating drugs on MSCs, John Shaughnessy from the Myeloma Institute for Research and Therapy at the University of Arkansas contacted us to see what we thought about the recent finding from his lab that Dkk1 levels are a good predictor of the extent of bone involvement in multiple myeloma. He thought that administration of MSCs into osteolytic lesions might allow repair of the lesions, once the osteoclasts had been inhibited with bisphosphonates. Based on reports from Andreef et al. (year) that MSCs support the growth of tumors, I didn’t think that adding more stromal cells into the area would be a good idea, and I subsequently found that conditioned medium from MSCs supports the growth of myeloma cells. I was able to confirm that conditioned medium from myeloma cells not only inhibits osteogenesis, but also induces MSCs to produce more IL6, the major growth factor for myeloma cells. Adding anti-IL6 antibodies to MSC conditioned medium blocked some, but not all, of the growth-promoting effect of the medium. With this in mind, administration of MSCs as is to a tumor-altered microenvironment didn’t seem like the best idea, and I decided to focus on repairing the tumor-induced alteration of the endogenous MSC microenvironment.

We first wanted to see if we could get an anabolic effect on bone in vivo from administration of drugs that modulate WNT signaling in MSCs, the presumptive precursor of osteoblasts in vivo. Lithium ions and bromoindirubin-3′-monooxime (BIO) both inhibit GSK3ß and therefore promote WNT signaling. For this reason, they were the first to be tested. The drugs did not lead to an increase in bone mineral content of wild-type mice, but rather had had unpredictable effects on bone mineral density. There was an effect on bone mineral content, but it was more of an expansion of the natural distribution of phenotypes, rather than a consistent increase. In a mouse model of osteogenesis imperfecta, however, the drugs did have an anabolic effect, consistent with the in vitro findings that normal homeostatic bone metabolism can’t be affected by WNT, but a defect can be corrected. At this point, I began to develop a mouse model of multiple myeloma in which we could test the drugs further for their effects bone, and also to see if repairing the microenvironmental defect produced a double effect on tumor growth by favoring the bone repair function of MSCs over their stromal support activity.

Design and Characterization of a Mouse Model of Multiple Myeloma

As I began to design the in vitro studies of MSCs and MM, there were two types of mouse models used to study MM. The most commonly used model called for subcutaneous administration of the cancer cells to immunodeficient mice. This model works well for the study of agents which work directly on the cancer cells, and allows the effects to be easily assessed by simply measuring the diameter of the tumor. However, the model does not recapitulate any of the systemic effects of myeloma, including the bone effects I was looking for. There is a model that does recapitulate some of the bone marrow engraftment and other features, but it requires implantation of human fetal bone tissue into scid mice, and our access to that tissue is limited. Some other models used IV administration of the cells into irradiated recipients, but our first attempt at that model resulted in 100% of the animals dying within 3 weeks, possibly from radiation sickness or related complications, despite our using a very low dose of radiation, or possibly due to cell aggregate-induced stroke. In order to maximize the chance of getting bone marrow infiltration by myeloma, while minimizing the loss of animals due to experimental injury, I developed a method for injection of cells directly into the marrow cavity of the tibia. This method allowed the myeloma cells to engraft and form tumors while not circulating around the bloodstream where they would form ectopic and less interesting but more fatal tumors. The progression of the tumor could be seen and measured as with the subcutaneous model, and bone effects could also be easily followed by xray of the injected leg. The contralateral leg also provided a convenient control for assessment of clinical parameters of bone metabolism, such as bone mineral density and trabecular volume. Out of 20 injected animals, 10 developed tumors on the injected leg. One of the animals was found to have large numbers of human CD138 positive cells in the bone marrow. I found elevated levels of calcium and N-telopeptides in the 10 animals, and N-telopeptides, but not calcium, was positively correlated with Dkk1 levels. Interestingly, there was no correlation between Dkk1 levels and tumor size. We initially were surprised by this result, but upon consideration it seems that the degree of vascularization, and the number of metabolically-active cells per tumor, is probably a bigger influence on serum levels than tumor volume.

Experiments in Progress

Measurements of trabecular volume and osteoclast to osteoblast ratio are in progress. ELISAs for free lambda chains, secreted by the tumor and for mouse Osteoprotegerin are also in progress. I have done differential counts on the mice, and will be looking to see if the degree of immune system impairment is correlated with any of the previous markers of tumor burden. Finally, the experiment will be repeated to determine the effect of BIO on the progression and severity of the tumors that develop in this model. One interesting experiment would be to see if the gene expression pattern of mouse MSCs in this model is similar to the gene expression pattern of MSCs derived from myeloma patients, and if BIO treatment affects this pattern.

References

1. Friedenstein, A. J., Gorskaga, U., and Kalungina, N. N. (1976) Exp. Hematol. 4, 267-274[Medline]
2. Gregory, C. A., Singh, H., Perry, A. S. & Prockop, D. J. (2003) J. Biol. Chem. 278, 28067-28078.[Abstract]
3. Tian E, Zhan F, Walker R et al. (2003) The role of the Wnt-signaling antagonist DKK1 in the development of osteolytic lesions in multiple myeloma. N Engl J Med 349,2483–2494.[Abstract]
4. Gunn, W. G., Conley, A., Deininger, L., Olson, S. D., Prockop, D. J., Gregory, C. A., (2005)Stem Cells 24,986-991. [Abstract]