**Angiogenic Potential of Bone Marrow Derived CD133**<sup>+</sup> **and CD271**<sup>+</sup> **Intramyocardial Stem Cell Trans-Plantation Post MI**

**Sarah Sasse 1,**†**, Anna Skorska 1,2,**†**, Cornelia Aquilina Lux 1, Gustav Steinho**ff **1,2, Robert David 1,2,\* and Ralf Gaebel 1,2**


Received: 6 October 2019; Accepted: 24 December 2019; Published: 27 December 2019

**Abstract:** Background: Bone marrow (BM)-derived stem cells with their various functions and characteristics have become a well-recognized source for the cell-based therapies. However, knowledge on their therapeutic potential and the shortage for a cross-link between distinct BM-derived stem cells, primed after the onset of myocardial infarction (MI), seems to be still rudimentary. Therefore, the post-examination of the therapeutic characteristics of such primed hematopoietic CD133<sup>+</sup> and mesenchymal CD271<sup>+</sup> stem cells was the object of the present study. Methods and Results: The effects of respective CD133<sup>+</sup> and CD271<sup>+</sup> mononuclear cells alone as well as in the co-culture model have been explored with focus on their angiogenic potential. The phenotypic analysis revealed a small percentage of isolated cells expressing both surface markers. Moreover, target stem cells isolated with our standardized immunomagnetic isolation procedure did not show any negative alterations following BM storage in regard to cell numbers and/or quality. In vitro network formation relied predominantly on CD271<sup>+</sup> stem cells when compared with single CD133<sup>+</sup> culture. Interestingly, CD133<sup>+</sup> cells contributed in the tube formation, only if they were cultivated in combination with CD271<sup>+</sup> cells. Additional to the in vitro examination, therapeutic effects of the primed stem cells were investigated 48 h post MI in a murine model. Hence, we have found a lower expression of transforming growth factor βeta 3 (TGFβ3) as well as an increase of the proangiogenic factors after CD133<sup>+</sup> cell treatment in contrast to CD271<sup>+</sup> cell treatment. On the other hand, the CD271<sup>+</sup> cell therapy led to a lower expression of the inflammatory cytokines. Conclusion: The interactions between CD271<sup>+</sup> and CD133<sup>+</sup> subpopulations the extent to which the combination may enhance cardiac regeneration has still not been investigated so far. We expect that the multiple characteristics and various regenerative effects of CD271<sup>+</sup> cells alone as well as in combination with CD133<sup>+</sup> will result in an improved therapeutic impact on ischemic heart disease.

**Keywords:** bone marrow stem cells; angiogenesis; myocardial infarction; cardiac regeneration

#### **1. Introduction**

In order to cover up the deficit of regenerative potential, new treatment approaches for cardiac diseases are needed. Several studies suggest that approaches based on the application of stem cells may be the future strategy in order to compensate the regenerative deficiency of the heart. As myocardial infarction (MI) occurs due to the absent blood flow in the coronary arteries, the formation of new blood vessels would be an essential prerequisite for the regeneration. Yet, in the search for suitable stem

cell subtypes, various other properties are of interest as well. This includes cells that interfere with pathological immune mechanisms as well as cellular mediators and chemokines that cause migration and homing processes. Besides, it is of the tremendous importance whether the cells are proofed to be safe in application. Furthermore, the cells can also be treated in advance in order to develop desirable properties. As the CD133<sup>+</sup> stem cell therapies and other approaches with hematopoietic cells provide promising results, research has to examine more cell types in order to achieve additional effects and thus to further improve the therapeutic measures. Until now, no stem cell-related cardiac complications following intramyocardial transplantation of bone marrow (BM) derived stem cells have been reported so that the therapy is considered to be safe [1]. In addition, the BM also contains a population named after their ability to differentiate into various cell types of mesenchymal origin. These multipotent stem cells are expandable and modifiable in cell culture and offer several distinctive characteristics, which make them a promising therapeutic approach for damaged heart tissue [2]. Especially mesenchymal stem cells (MSC) are able to decrease infarct size and myocardial fibrosis [3]. Thereby, cardiac remodeling in total may be attenuated as well as the heart's contractility improves [3]. Furthermore, MSC have paracrine effects that inhibit apoptosis of endothelial cells in hypoxic condition, increase their survival, and thus stimulate angiogenesis processes [4]. As another advantage, MSC do not express any antigens of histocompatibility and thus can be used in allogenic transplantation as well. Quite the contrary, these cells even have immunomodulatory paracrine effects that counteract inflammatory responses [5,6]. As inflammatory processes play a decisive role in the development of chronic heart diseases and have a negative influence on the heart function, these effects may also improve the therapy significantly. After the transplantation, MSC may be able to stimulate the production of reparative growth factors, inhibit local inflammatory response, and replace damaged cells [7]. Apart from these positive effects, difficulties with the practical application arise from the large heterogeneity of MSC as well. Thus, it is uncertain to predict which exact cell type arises from the MSC population after the application. Indeed, a study by Yoon et al. showed that the direct transplantation of unselected BM cells into the infarcted myocardium induces significant intramyocardial calcification [8]. This clearly demonstrates the research demands in order to guarantee the safety of MSC as therapeutic option. The development of methods for the identification and isolation of MSC subpopulations is crucial for a determined specific function and a successful clinical application. A currently much examined MSC subtype is characterized by the expression of the surface marker CD271. Several studies showed that this marker is one of the most specific concerning BM derived MSC [9]. Thus, there may be a particular suitability of CD271<sup>+</sup> MSC for preselected cell products. CD271 enables prospective cell isolation of MSC from BM derived mononuclear cells (MNC) [10] and have been shown to be essential in the formation of the heart as they influence the survival of endothelial cells, vascular smooth muscle cells, and cardiac myocytes. Furthermore, these proteins regulate angiogenesis and vasculogenesis by autocrine and paracrine mechanisms [10–12].

Knowledge of the therapeutic potential attributable to distinct BM derived stem cell subtypes primed due to MI of the donors is still greatly lacking. Therefore, this study intends to examine the benefits of CD271<sup>+</sup> and CD133<sup>+</sup> stem cells. In this context, we have explored the effects of CD271<sup>+</sup> cells alone as well as in combination with CD133<sup>+</sup> with the main focus on their angiogenic potential (in vitro examination). Moreover, the therapeutic capacities of both primed BM stem cells were investigated following cardiac ischemia/reperfusion in mice.

#### **2. Materials and Methods**

#### *2.1. Bone Marrow Aspiration*

Informed donors gave written consent to the aspiration of their BM according to the Declaration of Helsinki. The ethical committee of the University of Rostock has approved the presented study (registered as no. A201023) as of 29 April, 2010. BM samples were obtained by sternal aspiration from patients undergoing coronary artery bypass grafting (CABG) surgery at Rostock University, Germany. Anticoagulation was achieved by heparinization with 250 i.E./mL sodium heparine (B. Braun Melsungen AG, Melsungen Germany).
