(l to r) Biagio Saitta, PhD, associate professor, Department of Medicine, UMDNJ-Robert Wood Johnson Medical School, Camden; Vladimir Markov, MD, research associate; Shirin Modarai, MS, laboratory technician
Regenerative Medicine
T
|
he evolving field of “regenerative medicine” utilizes stem cells as a strategy to repair damaged tissue and preserve or regain function. Stem cells can be isolated from either adult or embryonic tissue and differ in their capacity to differentiate into multiple cell lineages. Mesenchymal stem cells (MSCs) are known to migrate to some tissues, particularly when injured or under pathological conditions, making these cells attractive for a number of therapeutic applications. Our research team has focused on isolating, characterizing and propagating MSCs from low volumes of umbilical cord blood (UCB), and on understanding molecular mechanisms involved in tissue repair of these adult stem cells.
Adult MSCs are multipotent, fibroblast-like cells that were first described in the mid-1970s, and were found in bone marrow as precursors of non-hematopoietic tissue. Cultured bone marrow MSCs have been transplanted in children with osteogenesis imperfecta (OI), a disease causing bone fractures and fragility. Reduced bone fractures and increased bone density were reported to be found when MSCs were engrafted into the defective bone. MSCs have also been isolated from other adult tissues, including adipose tissue, placenta, cord stroma, peripheral blood, and UCB. We obtained UCB samples from the New Jersey Cord Blood Bank (NJCBB), a facility housed within the Coriell Institute, where stem cell-rich cord blood is collected and distributed for transplantation and research. UCB is collected at birth from male and female infants from a varied ethnic population, whose parents have provided informed consent prior to donation. MSCs in culture dishes form adherent colonies, are self-renewing, and can differentiate into mesodermally-derived tissues, including cartilage, bone, fat and muscle. We were able to identify distinct types of genetically identical MSCs from UCB and comprehensively characterize their gene expression profiles. In collaboration with Dr. Kenro Kusumi at Children's Hospital of Philadelphia, these UCB-MSC profiles were compared to bone marrow-derived MSCs (Figure 1). This novel approach identified unique markers for a stem cell type with higher growth kinetics and colony-forming ability, and helped clarify the heterogeneity observed in these cells.
Since MSCs can differentiate into several cell types, and have less immune-related issues, they have been used in a number of preclinical and clinical trials, including treatment for cardiac infarct. Myocardial infarction (MI), or heart attack, is a major cause of morbidity and mortality worldwide. Each year, about 1.1 million Americans suffer from an acute MI, causing an average of 460,000 deaths per year, according to the American Heart Association. Heart attacks occur when heart muscle is deprived of oxygen. Deprivation results from blockage in blood vessels (coronaries), components normally supplying the heart with blood. Heart tissue responds to damage by scarring and fibrosis, resulting in loss of function, and often limits a person’s quality of life. Transplant and grafts are possible for many cardiac conditions, but donors are limited. MSCs have been shown, in animal models of heart attack, to engraft at the damaged site(s), attenuate pathologic remodeling of the heart tissue and reduce scar size, leading to improved post-MI cardiac function.
Figure 1. Morphology of distinct umbilical cord blood (UCB) derived mesenchymal stem cells (MSCs) and microarray expression analysis of UCB1, UCB2 and bone marrow-MSCs. Left panels. Phase contrast microscopy images of the UCB1 (A) and UCB2 (B) MSC populations isolated from the same UCB unit are shown. Magnifications are 40x and 200x. Right panels. A Venn diagram is shown, representing the distribution of 2,085 gene probe sets on a human gene expression microarray (HG-U133A). (B) Heatmap display of mean-centered and standardized gene expression patterns. The patterns were detected by hierarchical clustering of 290 probe sets differentially expressed between UCB1, UCB2 and BM-MSCs. The relative levels of gene expression are depicted with a color scale where red represents the lowest level, and green, the highest level, of expression (Modified from Markov et al., 2007).
With support from the New Jersey Commission on Science and Technology (NJCST), we investigated the ability of UCB-derived MSCs to respond to myocardial damage in an in vitro model. To simulate the effects of myocardial infarction at the molecular level, we analyzed injured rat cardiac cells (myocytes and fibroblasts) for apoptosis, necrosis and viability. We also identified specific extracellular matrix (ECM) and angiogenic genes expressed by the UCB-MSCs that are modulated when exposed to the hypoxic adult rat cardiac cells. Of these specific genes, the matrix metalloproteinases (MMP-1, MMP-2) and tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2) are involved in remodeling of injured and fibrotic tissues. Other genes involved in vascular remodeling of injured and fibrotic tissues include: angiopoietins (ANGPT1 and ANGPT2), chemokines (CXCL1 and CXCL6), fibroblast growth factors (FGF1 and FGF2), and interleukins (IL6 and IL8). These genes encode critical ECM and angiogenic proteins that provide a substrate for cells to migrate, grow and differentiate. As such, the matrix is an integral regulator of cell and tissue function. Our preliminary data shows increased expression of genes involved in the synthesis and remodeling of cardiac tissue and supports our aim of finding potential mechanisms involved in the process of cardiac repair. Through a collaborative effort with Drs. Steven Hollenberg and Joseph Parrillo, clinicians and cardiovascular researchers at Cooper Heart Institute, these studies are being extended to understand functional outcomes of our in vitro model. This approach will determine if MSCs can improve the contractility of the injured cardiac cells.
Figure 2. Engraftment of UCB-MSCs into a heart and rat tibialis anterior muscle.
(A) Left panels (top) show UCB-MSC-containing biogel transplanted into the ventricle. Middle
panels demonstrate engraftment of UCB-MSCs into ventricular wall (black arrows). Red
arrowheads show vessel-like structures. (B) Right panel, show human UCB-MSCs in myofibers after 14 days of transplant stained positive (orange) for the myogenic marker, Myf-5; DAPI was used for nuclear staining (blue); (100x magnification).
Our laboratory has initiated several collaborative projects, on both a national and international level, aimed at strengthening the stem cell biology program at Coriell. These collaborative projects include: i) studying the engraftment of our UCB-MSCs into the ventricles of a rat model of MI. With Dr. Marisa Jaconi, University of Geneva, Switzerland, we demonstrated that the cells invade and engraft into the myocardium and ventricular wall in the region of infarct and form vessel-like structures at 4 weeks post-infarct. Markers indicated that these structures originated from the transplanted human UCB-MSCs (Figure 2A), suggesting that MSCs differentiate toward endothelial lineages and may contribute to new blood vessels in the damaged tissue; ii) testing the potential of UCB-MSCs toward myogenic lineage differentiation in an in vivo model of skeletal muscle regeneration using a population of UCB-MSCs as a source for in vivo muscle repair. Our initial work, in collaboration with Drs. Parnigotto and Conconi, University of Padova, Italy, found muscular engraftment and myogenic differentiation, without immuno-rejection, of these stem cells after muscle injury (Figure 2B); iii) investigations with Drs. Margaret Keller and Jay Leonard, Coriell Institute, and Dr. Robert Nagele, UMDNJ-School of Osteopathic Medicine, to examine the safety of cord blood MSCs for cellular therapeutics by examining their stability after being grown in the laboratory (also featured in this issue).
Biagio Saitta received his PhD in biological sciences from the University of Messina, Italy. He was subsequently a postdoctoral fellow and junior faculty member at Thomas Jefferson University, Philadelphia. Dr. Saitta joined the Coriell Institute in 2002 as an associate professor and director of the Laboratory of Stem Cell and Matrix Biology, and has a faculty appointment in the Department of Medicine, UMDNJ-Robert Wood Johnson Medical School.
