NRSA Institutional Postdoctoral Training Grant (T32) at the

University of Louisville, James Graham Brown Cancer Center

“Current Trends in Stem Cell Therapies”.

(2017 – 2022)

PDs/PIs: Mariusz Ratajczak (Contact), M.D., Ph.D., D.Sci. / Sham S. Kakar, Ph.D., MBA

Regenerative medicine has become one of the most important scientific subjects and includes i) the physiology of tissue rejuvenation, ii) organ regeneration after injury and iii) the regulation of lifespan. Therefore, one of the most important educational challenges in this training program, is to train young investigators to become oriented and properly qualified stem cell scientists who can innovate and push forward these areas of research and explore novel translational applications. The main scientific focus of this “Current Trends in Stem Cell Therapies” training program is on stem cells isolated from adult tissues as well as on induced pluripotent stem cells (iPSCs) generated in the lab from postnatal tissues.

Taking the advantage of the strength of stem cell research at University of Louisville (UofL), the post-doctoral fellows will be involved in conceptual and laboratory work aimed at the optimization of i) isolation of versatile populations of stem cells from adult issues, ii) their ex vivo expansion alone or in combination with synthetic scaffolds iii) studies on the role of paracrine-related effects of stem cells in regeneration, with special emphasis on biological effects of extracellular microvesicles (ExMV), iv) strategies for stem cell delivery to damaged organs including to understand better mechanism orchestrating their homing and engraftment. These goals will be achieved with a multidisciplinary approach involving participating departments, centers, institutes, schools, and members of the training faculty at the UofL. This objective will be achieved by strong program and leadership with extensive experience in successful administration of the T32 training program; motivated and engaged faculty mentors selected from various academic units at UofL, and from various scientific disciplines related to stem cell biology, regenerative medicine; a well-designed and executed training program; an exceptional research and clinical environment with cutting-edge facilities; and scientific programs with national/international reputations for breakthrough discoveries in basic and clinical stem cell research.

Current challenges in stem cell therapies and regenerative medicine – relevant to this T-32 application.There are several challenging areas where significant improvement is needed for better clinical application of stem cells. Some of them are listed below. 

-To identify ethically accepted and therapeutically efficient stem cell for regenerative medicine. The only stem cells so far successfully employed in regenerative medicine are stem cells isolated from the adult tissues. It has been almost 50 years since the first hematopoietic transplants were introduced into the clinic based on application of hematopoietic stem cells (HSCs) isolated from bone marrow (BM), and subsequently from mobilized peripheral blood (mPB) and umbilical cord blood (UCB). Beside HSCs there are also attempts to employ other types of stem cells isolated from adult tissues e.g., mesenchymal stem cells (MSCs) isolated from BM, adipose tissue or umbilical cord, cardiac stem cells (CSCs) as well as expanded ex vivo epidermal progenitor cells. The first clinical trial employing CSCs was performed at UofL in the Department of Cardiology. In addition to well defined stem cells isolated from postnatal tissues, accumulating evidence reveals that adult tissues may also contain some other stem cell types endowed with broader trans-germ layer differentiating potential that could be specified to more than one germ line. It is well known that the stem cell compartment displays a developmental hierarchy. Some of these cells (e.g., very small embryonic like stem cells) express several markers characteristic of embryonic stem cells (ESCs).

- To understand therapeutic effects of adult stem cell therapies in regenerative medicine and explore practical utilization of stem cell-derived paracrine effects. Evidence has accumulated indicating that the beneficial effects of adult stem cell therapies in treatment of damaged organs is not because of trans-dedifferentiation of adult stem cells (e.g., HSCs becoming CSCs), but due to their paracrine effects (Figure 1A). Accordingly, adult stem cells (e.g., HSCs, MSCs, CSCs) secrete several soluble factors including i) peptide-based growth factors, cytokines, chemokines and enzymes that direct cell responses and modify surrounding microenvironment (e.g., metalloproteinases, enzymes processing extracellular ligands), ii) bioactive lipids (e.g., sphingosine-1-phosphate, creamide-1 phosphate, lipophosphadic acid, eicosanoids) and iii) extracellular nucleotides (e.g., ATP, UTP). All these soluble paracrine factors play an important role in interaction between cells. In parallel, growing attention has recently focused on cell-to-cell communication that involves paracrine effects of cell-derived spherical membrane fragments called extracellular macrovesicles (ExMVs), a mechanism that for many years has been largely ignored and overlooked. Accordingly, both soluble factors as well as non-soluble ExMVs if released from the cells employed as cellular therapeutics in regenerative medicine seem to play an important role in improving the function of damaged organs. They may i) inhibit apoptosis of cells residing in the damaged tissues, ii) stimulate proliferation of cells that survived organ injury, and iii) stimulate vascularization of affected tissues. Post-doctoral trainees will be studying these mechanisms and to assist in optimizing them to improve cellular therapies (e.g., by developing and engineering more efficient therapeutically ExMVs) (Figure 1B).

Figure 1. Panel A - Using a heart infarct model, two possible scenarios illustrating the beneficial effects of stem cell therapies in regenerative medicine. Left panel (1^st scenario). Cells employed for therapy (e.g., HSCs or MSCs) may theoretically transdifferentiate into cardiomyocytes. However, if this occurs at all, it is a very rare and random phenomenon and is not well substantiated by current experimental data. Right panel (2^nd scenario). Cells employed for therapy (e.g., HSCs or MSCs) do not transdifferentiate into cardiomyocytes, but secrete several paracrine factors and shed MVs that inhibit apoptosis in damaged cardiomyocytes, promoting their proliferation and stimulating angiogenesis. Evidence is accumulating that this is a major effect in currently employed stem cell therapies. Panel B. Different approaches to generating more efficient pro-regenerative MVs in vitro. MVs could be harvested from large-scale in vitro cultures of MV-producing cell lines. Such cell lines may be modified to obtain MVs that i) do not express HLA antigens, ii) are enriched in growth factors, cytokines, and chemokines that promote regeneration of damaged organs, iii) are enriched in mRNA and regulatory miRNA facilitating regeneration of damaged tissues and/or promoting angiogenesis, and iv) display molecules that direct them to, and subsequently retain them in, damaged tissues.

- To understand better mechanisms that govern stem cell self-renewal, differentiation and aging.  Mechanisms of self-renewal and maintenance of stem cell are not fully understood. Several of the investigators at UofL who are members of this T-32 training program are interested in molecular mechanisms that govern stem cell fate. The crucial problem in stem cell expansion strategies is to prevent increase of population of progenitor cells due to depletion of stem cell pool. Therefore, it is important to identify genes that are playing a role in physiological stem cell renewal based on symmetric stem cell division to preserve a pool of stem cells during expansion.

- To optimize stem cell delivery, homing, engraftment and stem cell isolation and mobilization strategies. Stem cell delivery by systemic infusion or local injection depends on homing and engraftment of stem cells. Homing of HSCs to their niches precedes engraftment and the establishment of transplant-derived hematopoiesis. It is enforced by chemotactic factors released in a given microenvironment that attract migrating cells. These chemotactic factors may be peptide-based (e.g., chemokines or growth factors), bioactive phosphosphingolipids (e.g., S1P or C1P), or extracellular nucleotides (e.g., ATP or UTP). Stem cells may also respond to a Ca²⁺ or H⁺ gradient by employing calcium- or proton-sensing receptors, respectively. Homing may also be sensitized by certain factors, such as small antimicrobial cationic peptides (e.g., LL-37 or b2-defensin), or modulated by certain pharmacological compounds that affect cholesterol-enriched membrane lipid raft formation. Homing can also be understood more broadly as the migration of non-hematopoietic stem cells to specific areas in tissues when other types of stem cells navigate in the tissues by sensing chemotactic gradients that direct them to lodge in new locations.

Faculty Members at T32 Training Grant

For detail information and availability of post-doctoral position, please contact:

Mariusz Z. Ratajczak, M.D., Ph.D., Dsc.
Professor
Department of Medicine, James Graham Brown Cancer Center
mzrata01@.louisville.edu

Sham S. Kakar, Ph.D., MBA
Professor
Department of Physiology, James Graham Brown Cancer Center
sskaka01@louisville.edu