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Reference:  Yamada Y, Yokoyama S-I, Wang Z-D, Fukuda N, Takakura N. Cardiac stem cells in brown adipose tissue express CD133 and induce bone marrow nonhematopoietic cells to differentiate into cardiomyocytes.

Summarized by: Tara Gooen and Edward Vallejo, Fall 2007


LAY REVIEW

Introduction

Researchers have been working on a way to repair damaged cardiac tissue after a heart attack. Altering genes and transplanting stem cells into the affected area are two methods that show the most promise of working. This paper focuses on a group of newborn fat cells which could be a source of cardiac cells that could be used for the repair process. The authors also see whether mesenchymal (bone) stem cells when compared with the hematopoietic (blood and immune) stem cells are better at becoming these cardiac cells.


The cardiac cells were formed by growing two different types of cells together. To show that they were not formed because of fusion between the two different cell types, isolated and cultured bone marrow cells, including both mesenchymal and hematopoietic stem cells, were injected into the hearts of mice that had just suffered a chemically induced heart attack. 

 

Surface Characteristics of Possible Cardiac Stem Cells in Fat Tissue

Fat tissue was removed from unborn and newborn mice and the fat cells were analyzed for three cell surface markers (CD45, Ter119, and CD31) by flow cytometry, a cell sorting technique. Cells that did not express those markers were further examined for expression of three other markers, (c-kit, Sca-1, and CD133). Cardiac cells were then formed from each of these cell populations and it was observed that those cells that positively expressed the CD133 marker were the best at becoming the cardiac cells because they possessed cardiac markers on their surface. Studies involving heart drugs also showed that these cells not only had the surface properties, but also the functional capability of cardiac cells since they responded just like cardiac cells would to those drugs. 

 

Newborn Fat Cells Cause Cardiac Cell Formation from Bone Marrow Cells

Previous studies have shown that in order for the bone marrow cells to spontaneously develop into cardiac cells, the right microenvironmental factors were needed. The authors’ experimental model achieved this by culturing bone marrow cells from one type of mice, whose cells expressed a green fluorescent protein when viewed under the microscope, with CD133+ newborn fat cells from mice that did not have this protein for 14 days. These two types of cells were directly touching one another and developed into cardiac cells.
 


Bone Marrow Stem Cells Cultured with Newborn Fat Cells Developed into Cardiac Cells without Fusing with Each Other

To show that these cardiac cells did not develop because of the bone marrow stem cells fusing with the newborn fat cells, the two were separated from each other by a 0.4 μm membrane. This physical barrier had small openings in it to allow soluble factors to pass between the two cells to mimic the microenvironment around the cells that would normally be present. The authors concluded that the newborn fat cells did not fuse with the newly formed cardiac cells. This was because cell signals that were only seen in each cell type before the beginning of the experiment were still observed at the same strength after 14 days. They also learned that the potential to form cardiac cells was not as strong in the indirect contact model as in the direct contact model.

 

Cell Contact Is Critical for the Converting Bone Marrow Stem Cells into Cardiac Cells

As the authors established in the previous section, bone marrow stem cells did not just fuse with the newborn fat cells to appear like cardiac cells. However, the efficiency of bone marrow stem cells converting into cardiac cells was only 20% when the bone marrow and newborn fat cells were not in direct contact.  The authors then chemically fixed the newborn fat cells to prevent any communication via soluble factors.  This did not stop the conversion. Therefore, the authors concluded that there might be two independent mechanisms in the conversion: (1) factors which the cells secrete that do not need direct contact between cells and (2) cell-to-cell surface communication which does require direct contact.  Further, the authors showed that an important calcium dependent cell-to-cell surface communication mechanism is instrumental in the conversion.

 

Newly Formed Cardiac Cells Contributed to Heart Regeneration

The authors then studied the usefulness of these cardiac cells in mice.  First, bone marrow stem cells were cultured with the newborn fat cells.  The bone marrow stem cells showed conversion factors after only 5 days.  The bone marrow cells were then isolated and cultured for another 14 days.  Those cells were injected into the hearts of mice which were suffering from a chemically induced attack.  The authors observed several improvements in the hearts of mice that received the cardiac cells, including the communication of these cardiac cells with the existing cardiac cells.

 

Mesenchymal Stem Cells in the Bone Marrow Are a Major Source of Newly Formed Cardiac Cells
 
Since bone marrow stem cells are a mixed population of cells, including mesenchymal (bone) stem cells and hematopoietic (blood and immune) stem cells, the authors studied which type of stem cell converted to cardiac cells better. It was found that there was a 20-fold difference in the conversion of mesenchymal stem cells when compared with the conversion of hematopoietic stem cell. 

 

Summary


The paper further demonstrates that the authors may have discovered a source of cardiac stem cells. However, the use of a mixed population of bone marrow stem cells raises unanswered questions. We would like to see similar experiments performed with only mesenchymal stem cells, to study the efficiency of conversion and what specific factors are necessary. In this paper, the authors have further shown the possibility of CD133+ newborn fat cells as being a source of cardiac stem cells. There are many possibilities for further research.

 

SCIENTIFIC REVIEW

Introduction

The repair of damaged cardiac tissue following myocardial infarction to the level of viable functionality has been the goal of researchers in recent years.  Two important areas of investigation that show the most promise for generating positive results include genetic manipulation and stem cell transplantation. The primary focus of this paper is experimentation with a specific population of cells in Brown Adipose Tissue (BAT) that could most likely be a source of Cardiac Stem Cells (CSCs). Additional experiments also give evidence to support whether MSCs or HSCs from a bone marrow mixed population are better at differentiating into cardiomyocytes (CMs). Effective demonstration of an in vivo infarction model attempts to show that the mechanism of CM induction and repair through coculturing is not through cell fusion, but through bivalent cationmediated cell-to-cell contact between cells.

 

Surface Phenotype of CM Progenitors in BAT

Brown Adipose Tissue was dissected from neonatal and postnatal mice and analysis was carried out on the Brown Adipose Tissue Derived Cells (BATDCs) for three cell surface markers by flow cytometry (CD45, Ter119, and CD31). Among cells negative for the aforementioned markers, positive expression of the markers c-kit (1.4%), Sca-1 (16.6%), and CD133 (3.5%) was observed. From each of these respective populations, CMs were derived and analyzed using immunocytochemical analysis and transmission electromicrographs. Of these, CD133+ cells were shown to be the best at differentiating into CMs based on criteria such as surface adherence and positive expression of known CM markers such as SA, GATA, Troponin T and Troponin I.

In addition, pharmacological studies of this specific cell population show that they exhibit CM-like responses to medication in both an agonistic and antagonistic manner. Overall, these results provide evidence that not only do these differentiated cells express phenotypic markers of CMs, but they also possess their functional capabilities as well.

 

BATDCs Effectively Induce CM Production from BMCs

Previous in vitro studies showed that although Bone Marrow Cells (BMCs) were a source of CMs, they could not spontaneously differentiate into CMs. In order to produce these results, the appropriate microenvironmental cues were required. The experimental model accomplished this through 14 days of coculturing of Bone Marrow Mononuclear Cells (BMMNCs) from green fluorescent protein mice with CD133+ BATDCs from ROSA 26 wild-type mice. These two types of cells were in direct surface contact with one another and produced differentiated, surface adherent CMs.

 

BMMNCs with BATDCs Differentiated into CMs Without Fusion Mechanism

In order to show that CM development was not due to fusion between the BMMNCs and BATDCs, the two cell types were physically separated by a 0.4 μm membrane. This allowed the passage of microenvironmental cues such as cytokines between the cells while simultaneously maintaining a strict physical boundary. Signals distinct to both cell types were shown by RT-PCR to still be differentially expressed 14 days after coculturing. This led the authors to conclude that fusion did not play a role in CM differentiation. However, differentiation potential in the indirect coculturing model was not as efficient as it was in the direct contact experiments.

 

Cell Contact Mediated by Bivalent Cation Is Critical for the Differentiation of BMMNCs into CMs

As the authors established in the previous section, it was seen that cell fusion is not the only factor in the differentiation of BMMNCs into CMs. However the efficiency of differentiation from BMMNCs to CMs without direct contact coculture was only 20% of the differentiation when compared with with direct contact coculture between BATDCs and BMMNCs.  To further investigate the possibility of cell fusion as a major factor in the differentiation, the authors demonstrated that BMMNCs differentiated into CMs after chemically fixing the cell surface of the BATDCs with paraformaldehyde. 
Additionally, the investigators studied cadherin-mediated calcium dependent cell-to-cell contact in the regulation of BMMNC differentiation in CMs.  Through the use of calcium chelator EDTA or EGTA, the authors showed that all differentiation of BMMNCs to CMs is suppressed.  Further, via anti-Ecad antibody or soluble cadherin, the authors showed that Ecad-mediated cell-to-cell contact is also a key component in the differentiation of BMMNCs.  This suggests that there might be two independent ways of inducing CMs from BMMNCs: secreted factors and membrane proteins.


Educated BMMNCs Contributed to CM Regeneration
The authors then performed in vivo studies in murine models.  First, it was determined that BMMNCs showed a commitment into CMs after only 5 days of coculture culture with BATDCs when followed by 14 days of isolated culture.  About 9.7% of BMMNCs showed CM lineage markers.  The authors then injected these differentiated BMMNCs into the border of infected hearts of mice after the induction of an acute myocardial infarction.  They observed significant improvement in the mice that received the transplanted cells, such as the formation of gap junctions, intercalated disks, and secretions of cardiac myocyte-specific factor.  Further, the authors showed that the differentiated BMMNCs had not undergone cell fusion after implantation via FISH staining.


Nonhematopoietic Cells in the BM Are a Major Source of CM

To determine what type of cells are differentiating into CMs, the authors sorted the differentiated BMMNCs cells by lineage markers and determined that there was a 20-fold difference in the differentiation of nonhematopoietic stem cells (CD34-, CD31-, CD105+, mesenchymal) versus hematopoietic stem cells (Lin-, c-kit+).

 

Discussion

The paper further demonstrates, in conjunction with the previously published paper, that the authors may have discovered a source of cardiac stem cells.  While the cell fusion experiments are useful, the use of a mixed population of BMMNCs raises unanswered questions.  It would be nice to see the same experiments performed with only mesenchymal stem cells to determine the efficiency of differentiation and determine if there are additional factors in the mixed population that are not directly associated with BMMNCs.  Additionally, the determination of which factors the BATDCs actually provide to induce BMMNC differentiation would be very interesting.  In this paper, the authors have further shown the possibility of CD133+ BATDCs as being a source of cardiac stem cells.  There are many possibilities for further research.

 


 

ACKNOWLEDGEMENTS


This review was prepared by the following graduate students, Advanced Stem Cell Seminar (Fall 2007), University of Medicine and Dentistry of New Jersey, Graduate School of Biomedical Sciences:
Tara Gooen and Edward Vallejo
Course Instructor : Pranela Rameshwar, Ph.D.

 

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