User:Christina Katherine Bray/Notebook/Muscle Regeneration Using Ecto-MSCs

Project Overview

This project focuses on designing an assay with which to test the best conditions under which muscle-derived stem cells differentiate into the three different types of muscle tissue (skeletal, smooth, and cardiac). We aim to find the optimal path for muscle differentiation so that, after further development, this path can be used on muscle cells taken from a patient to differentiate them into the desired type of muscle tissue and then these cells can be implanted into the area with the muscle damage or atrophy. This research aims to address the social problem that many cases of muscle damage do not yet have good treatments; for example, patients with severe heart muscle damage often undergo invasive heart transplants and patients with skeletal muscle damage often go undiagnosed or untreated and the damage heals as scar tissue, often causing permanent pain or mobility limitations. On a scientific level, stem cells could not be induced to develop into functional muscle tissue until very recently, and there are still many problems with the approach that need to be dealt with in order for it to become a viable treatment for humans. These are the issues that our project seeks to address.

Background Information

This section is a work in progress.

Muscle-Derived Stem Cells offer distinct advantages over:

(1) Embryonic stem cells - MDSCs allow autologous therapy which minimizes risk of an immune response to the therapy.

(2) Bone marrow-derived stem cells - MDSCs don't necessitate the painful process of bone marrow extraction, requiring only a much less invasive procedure.

Research Problem and Goals

1) Inducing skeletal muscle cells to become stem cells (called skeletal muscle-derived stem cells or MDSCs).

2) Designing an assay through which to test the effect of compounds of interest on differentiation of the MDSCs into skeletal, smooth, and cardiac muscle; identifying the compounds of interest (often growth factors) through a literature search.

3) Using the assay to identify the compounds which most successfully induce differentiation, as measured by the expression of certain muscle-type specific proteins.

Project Details and Methods

This section is a work in progress.

Predicted Outcomes

The projected outcome of this study is the development of an assay that we will use for the identification of optimal paths of differentiation of MDSCs into skeletal, cardiac, and smooth muscle tissue. This assay could also be used, however, to test for degree of muscle differentiation from different types of stem cells to optimize this portion of the study. In later studies, other groups could utilize our assay design, but with different test compounds, to test for differentiation of stem cells into other types of tissue for regenerative treatment purposes.

Needed Resources

This section is a work in progress.

Potential Societal Impact

Ideally the results of this study would identify the best methods for treating muscle damage of all types (cardiac, skeletal, and smooth) in vitro in order to improve therapies for these serious health problems. The impact of this study could potentially be quite large; the development of a treatment for regenerating heart muscle alone could potentially improve the conditions of the approximately one million Americans who have heart attacks (which often lead to cardiac muscle atrophy) every year and the impacts from treatments for muscle damage of other muscle types only add to the number of people who could be helped by this research.

References

1) Xin Nie, Yongjun Xing, Manjin Deng, Li Gang, Rui Liu, Yongjie Zhang, Xiujie Wen. (2014) Ecto-Mesenchymal Stem Cells from Facial Process: Potential for Muscle Regeneration. Cell Biochemistry and Biophysics. http://link.springer.com/article/10.1007%2Fs12013-014-9964-x/fulltext.html

This study focuses on the differentiation of ecto-MSCs in regenerating muscle tissue. EMSCs help in the formation of teeth, salivary glands, and muscle tissue in early development, and thus it was hypothesized that the transplantation of EMSCs might induce them to differentiate into mature skeletal muscle. It was discovered that EMSCs, in mice and therefore potentially in humans, can accumulate and form myotubes and continue differentiating into skeletal muscle when in a sheet of cells in vitro, so there is potential for cell therapy and engineering tissue that can repair skeletal muscle.

2) Rui-feng Qin, Tian-qiu Mao, Xiao-ming Gu, Kai-jing Hu, Yan-pu Liu, Jin-wu Chen, Xin Nie. (2007) Regulation of skeletal muscle differentiation in fibroblasts by edogenous MyoD gene in vitro and in vivo. Molecular and Cellular Biochemistry. http://link.springer.com/article/10.1007%2Fs11010-007-9446-1

This study provided an analysis of how myogenic regulatory factors (MRFs), and specifically MyoD helps regulate skeletal muscle differentiation. NIH3T3 cell lines (embryonic mice fibroblasts) were transfected such that exogenous MyoD was expressed at high levels. When grown in vivo (in mice) and in vitro, the fibroblasts underwent myogenesis and remained stable, so it was concluded that MyoD may play an important role in cell-mediated gene therapy of skeletal muscle.

3) Stella Alimperti, Hui You, Teresa George, Sandeep K. Agarwal, Stelios T. Andreadis. (2014) Cadherin-11 regulates mesenchymal stem cell differentiation into smooth muscle cells and development of contractile function in vivo. Journal of Cell Science. http://jcs.biologists.org/content/early/2014/04/13/jcs.134833.long

MSC differentiation toward smooth muscle cell (SMC) can be induced by some soluble factors, such as Transform Growth Factor beta 1 (TGF-β1) but is also influenced by adherent junctions. This study found that Cadherin-11 but not Cadherin-2 was necessary for MSC differentiation into SMC by influencing TGF-β receptor II pathway as well as a Rho-associated protein kinase pathway and inducing contractile function both in vitro and in vivo.

4) Wei Huang, Din-Zhang Xiao, Yigang Wang, Zhi-Xin Shan, Xiao-Ying Liu, Qiu-Xiong Lin, Min Yang, Jian Zhuang, Yangxin Li, Xi-Yong Yu. (2014) Fn14 Promotes Differentiation of Human Mesenchymal Stem Cells into Heart Valvular Interstitial Cells by Phenotypic Characterization. Journal of Cellular Physiology. http://onlinelibrary.wiley.com/doi/10.1002/jcp.24480/abstract;jsessionid=15D39A090EF3CAEC4688DFE9AA258621.f03t01

This study focused on using bone marrow derived MSCs and vectors that over express the fibroblast inducible factor 14 (Fn14) gene in order to see if they could induce differentiation into cells used in heart valves. It was found that the expression of α-smooth muscle actin (SMA) was significantly higher with Fn14, and the phenotype of these in vitro cells were similar to the phenotype of normal heart valves, so this may provide a therapeutic strategy for heart valve disease treatment.

5) Jason Tchao, Jong Jin Kim, B. Lin, G. Salama, C.W. Lo, L. Yang, Kimimasa Tobita. (2013) Engineered Human Muscle Tissue from Skeletal Muscle Derived Stem Cells and Induced Pluripotent Stem Cell Derived Cardiac Cells. International Journal of Tissue Engineering. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3984572/#__ffn_sectitle

In this study, they experimented whether muscle differentiated from skeletal muscle derived stem cells (MDSCs) can express phenotypes (such as sarcomere proteins and transcription factors) shared between developing cardiac and skeletal muscle cells. They compared engineered muscle tissue derived from MDSCs to induced pluripotent stem cell-derived cardiac cells (iPS-EMT), and found that MDSCs can differentiate into muscle cells that mimic both developing cardiac and skeletal muscle which could possibly lead to therapeutic options of MDSCs in cardiac repair treatment.

6) Lin Wang, Lan Cao, Janet Shansky, Zheng Wang, David Mooney, Herman Vanderburgh. (2014) Minimally invasive approach to the repair of injured skeletal muscle with a shape-memory scaffold. Molecular Therapy. http://www.nature.com/mt/journal/vaop/naam/abs/mt201478a.html

Repairing injured skeletal muscle by cell therapy is a result of injected cells, but cell survival has typically been low. This study designed a degradable scaffold that can be implanted, in addition to the delivery of myoblasts and growth factors, that promoted the survival of cells in vivo and thus the functional regeneration of injured skeletal muscle.

7) Fiona C. Lewis, Beverly J. Henning, Giovanna Marazzi, David Sassoon, Georgina M. Ellison, Bernardo Nadal-Ginard. (2014). Porcine Skeletal Muscle-Derived Multipotent PW1pos/Pax7neg Interstitial Cells: Isolation, Characterization, and Long Term Culture. Stem Cells Translational Medicine. http://stemcellstm.alphamedpress.org/content/early/2014/04/17/sctm.2013-0174.long

This study isolated skeletal muscle-derived interstitial progenitor cells and started to explore the potential of these cells for use in tissue engineering and muscle regeneration from a single multipotent stem cell type. They show that these cells can give rise to skeletal myoblast/myotubes, smooth muscle, and cardiomyocyte-like cells, which indicates that skeletal muscle (an easily accessible source) may be able to help regenerate other types of muscle tissue.

8) Akira Ito, Yasunori Yamamoto, Masanori Sato, Kazushi Ikeda, Masahiro Yamamoto, Hideaki Fujita, Eiji Nagamori, Yoshinori Kawabe, Masamichi Kamihira. (2014). Induction of functional tissue-engineered skeletal muscle constructs by defined electrical stimulation. Scientific Reports. http://www.nature.com/srep/2014/140424/srep04781/full/srep04781.html

Because electrical impulses are necessary for skeletal muscle development in vivo, this study explored the idea of using electrical stimulation (and attempted to optimize a protocol) to fabricate functional skeletal muscle tissue in vitro, which may be used for therapies to restore damaged muscle.

9) Mark Juhas, George C. Engelmayr Jr., Andrew N. Fontanella, Gregory M. Palmer, Nenad Bursac. (2014). Biomimetic engineered muscle with capacity for vascular integration and functional maturation in vivo. PNAS. http://www.pnas.org/content/111/15/5508.long

This study describes the creation of biomimetic skeletal muscle tissues with phenotypes (structural/functional/myogenic properties) characteristic of native muscle tissue that also can be implanted in vivo for restoration. They also state that the implantation process is enhanced by formation of muscle architecture in vitro.

10) Toma C, Pittenger MF, Cahill KS, Byrne BJ, Kessler PD. Human Mesenchymal Stem Cells Differentiate to a Cardiomyocyte Phenotype in the Adult Murine Heart. Circulation 2002; 105: 93-98. https://circ.ahajournals.org/content/105/1/93.full

This study used human MSCs (retrieved from the bone marrow) and engrafted them in the myocardium of mice. The cells differentiated in cardiomycocytes (heart muscle cells) and expressed proteins (including those needed for contraction) levels similar to those of native cells.

11) Ota S, Uehara K, Nozaki M, Kobayashi T, Terada S, Tobita K, Fu FH, Huard J. Intramuscular Transplantation of Muscle-Derived Stem Cells Accelerates Skeletal Muscle Healing After Contusion Injury via Enhancement of Angiogenesis. Am J Sports Med 2011; 39: 1912-1922. http://ajs.sagepub.com/content/39/9/1912.full

This study aimed to determine the optimal time for the implantation of Muscle-derived stem cells (MDSCs) into muscle affected by contusion in order to minimize scar tissue and maximize regeneration. The researchers concluded that the ideal time frame for this transplantation was 4 days after the contusion.

12) Lepper C, Partridge TA, Fan C-M. An absolute requirement for Pax7-positive satellite cells in acute injury-induced skeletal muscle regeneration. Development 2011; 138: 3639-3646. http://dev.biologists.org/content/138/17/3639.full

This study concluded that Pax7 must be expressed in satellite cells in order for regenerative myogenesis to occur. This study indicates the critical nature of Pax7 to regeneration of damaged skeletal muscle tissue.

13) Le Grand F, Grifone R, Mourikis P, Houbron C, Gigaud C, Pujol J, Maillet M, Pagès G, Rudnicki M, Tajbakhsh S, Maire P. Six1 regulates stem cell repair potential and self-renewal during skeletal muscle regeneration. JCB 2012; 198: 815-832. http://jcb.rupress.org/content/198/5/815.full

This study found that the homeodomain transcription factor Six1 controlled transcription of MyoD, Myogenin, and Dusp6 which have already been linked to skeletal muscle regeneration. Therefore, the Six1 homeoproteins regulate both the regeneration of damaged skeletal muscle tissue and the maintenance of muscle stem cell populations.

14) Charge SBP, Rudnicki MA. Cellular and Molecular Regulation of Muscle Regeneration. APS 2004; 84: 209-238. http://physrev.physiology.org/content/84/1/209.full

This paper summarizes the differentiation of skeletal muscle in normal development and native regeneration. In development, the upregulation of the transcriptional activators MyoD and Myf5 is required for the differentiation of stem cells into muscle cells. The proteins Myogenin, MRF4, myosin heavy chain (MHC), and muscle creatine kinase (MCK) are produced later in embryonic development and are necessary for viable muscle tissue. Of muscle regeneration, the paper identifies these key goals: regeneration of the myofibers, revascularization, reinnervation, and reconstitution of the extracellular matrix. The paper also identifies that after an injury, muscle first undergoes degeneration, which stimulates regeneration. The paper identified many key factors that regulated muscle regeneration. Of particular interest are HGF and IGFs. Hepatocyte Growth Factor (HGF) promotes satellite cell differentiation and migration to site of injury. Insulin-like Growth Factors (IGFs) are implicated in satellite cell differentiation, cell hypertrophy (growth due to cytoplasma increase), and reinnervation.

Notes

• This research proposal is a class project for the Laboratory Fundamentals of Biological Engineering (20.109) class at the Massachusetts Institute of Technology taught in Spring 2014.