**Platelet Lysate to Promote Angiogenic Cell Therapies Platelet Lysate to Promote Angiogenic Cell Therapies**

Scott T. Robinson and Luke P. Brewster Scott T. Robinson and Luke P. Brewster

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66934

#### **Abstract**

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Cellular therapies for patients with ischemic muscle have been limited by poor cell reten‐ tion and survivability. Platelets are a robust source of growth factors and structural pro‐ teins, and extracts from this peripheral blood component may be manipulated to improve both cell retention and survivability in percutaneous delivery methods. Human platelet lysate is generated from pooled human platelets and contains a growth factor milieu that promotes robust human mesenchymal stem cell (MSC) proliferation without risk of xenogenic contamination. As such, platelet lysate is a practical alternative to animal serum for MSC culture and, with minor adjustments to the production process, can also be used as a scaffold for cell delivery. Human platelet lysate is a promising substrate that can provide nutritive delivery both in vitro and during cell implantation, potentially improving retention and survivability of MSCs that may improve angiogenic function for cell therapy in *treatment* of ischemic tissues.

**Keywords:** critical limb ischemia, mesenchymal stromal cells, platelet lysate, angiogenic cell therapy

## **1. Introduction**

The legs are a site of ischemic muscle that are particularly attractive to application of angio‐ genic therapies. Decreased perfusion of the legs is known as peripheral arterial disease (PAD), and PAD is pandemic with extreme costs to society. In its most severe form, it is called critical limb ischemia (CLI). CLI patients do not have adequate perfusion to their resting nutritional needs resulting in rest pain or even tissue loss. While only 1–2% of PAD patients will develop CLI, CLI affects 1 million individuals annually [1]. Surgical revascularization of CLI patients can prevent major amputation. Current treatment for limb salvage includes endovascular therapy (angioplasty or stent placement) and surgical bypass. However, despite improve‐ ments in medical and surgical therapies, successful management of CLI is difficult with ∼0.5

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

of patients with CLI dying or undergoing major amputation within 1 year [2]. Since many patients with CLI either fail revascularization therapy or are not candidates for these proce‐ dures, amputation is commonly performed. In addition to the impaired quality of life that results from lower limb amputation, historically mortality rates for this procedure have been reported to approach 40% [3]. New and innovative therapies are imperative to improve mor‐ tality and quality of life in patients with CLI and provide an alternative to limb amputation.

Cell therapy is a promising new treatment strategy for patient with CLI. The concept of cell therapy for treatment of critical limb ischemia coincided with the discovery of a circulat‐ ing endothelial progenitor cell (EPC) in 1997, which described a population of circulating peripheral blood mononuclear cells that presumably arise from the bone marrow yet is also capable of displaying characteristics of a mature endothelium [4]. This discovery challenged the prevailing paradigm of postnatal neovascularization [5] in which blood vessel growth was the result of angiogenesis (the formation of new blood vessels from the existing endothe‐ lium through sprouting or intussusception) and arteriogenesis (the expansion of preexisting collateral vessels due to an increase in blood flow in response to changes in shear stress [6]). The work by Asahara et al. was significant for two reasons. First, it introduced the concept of postnatal vasculogenesis, suggesting that bone marrow‐derived cells could contribute to the formation of new blood vessels in the adult similar to the process of vasculogenesis seen in embryogenesis, where primitive hemangioblasts give rise to de novo blood vessels in the developing fetus. Second, it pioneered the idea of cell therapy, through experiments in which the ischemic limbs of nude mice were injected with a population of human peripheral blood mononuclear cells enriched for CD34+ and Flk‐1 (VEGFR‐2), and found that the transplanted cells incorporated into the blood vessel endothelium at sites of neovascularization.

The notion that bone marrow‐derived cells incorporate directly into native endothelium is controversial. Despite several initial reports suggesting differentiation of bone marrow cells into endothelial cells [7–9], several subsequent studies demonstrated that endogenous and transplanted bone marrow cells did not directly incorporate into the endothelium but instead support neovascularization through a paracrine mechanism [10–13]. Numerous stem and pro‐ genitor cell populations derived from the bone marrow, adipose tissue, and embryonic stem cells and induced pluripotent stem cells have been shown to enhance blood vessel growth in animal models of hind limb ischemia, suggesting a role for cell therapy in therapeutic neovas‐ cularization [4, 14–16]. Mesenchymal stromal cells (MSCs) are a potential cell source that can be easily isolated, rapidly expanded ex vivo, and have potent proangiogenic qualities [17] medi‐ ated through paracrine stimulation of endogenous tissues [18]. The use of MSCs for cell therapy is advantageous over other cell types because they can be derived from an autologous source, thereby avoiding the immunogenicity and loss of tolerance observed when allogeneic MSCs are exposed to an inflammatory environment [19, 20].

Despite numerous animal studies and small clinical pilot trials demonstrating the ability of MSCs to promote angiogenesis [17, 21, 22], the biology and clinical benefit of this approach has not yet been demonstrated in patients with CLI. Two major limitations have prevented the translation of MSC therapy from the laboratory to the clinical arena. First, the use of an autolo‐ gous cell source in this demographic is complicated by the fact that progenitor cell populations are depleted or functionally impaired in patients with coronary artery disease [23], stroke [24], and diabetes mellitus [25–27] and who are smokers [28]; all risk factors or comorbidi‐ ties are highly prevalent in patients with CLI. A second major limitation of stem cell therapy thus far has been maintaining a clinically significant cell number in target tissues, as direct intramuscular injection or intra‐arterial infusion alone typically does not enable adequate cell delivery [29–31]. In order to fully realize the potential of cell therapy, these limitations must be addressed before successful clinical deployment of MSC therapy can be achieved.

As cell therapy is translated from preclinical animal studies to human clinical trials, strict cell culture techniques must be employed to ensure human safety. The vast majority of clinical tri‐ als to date have utilized fetal bovine serum (FBS) for ex vivo expansion and growth of MSCs. However, FBS has considerable xenogenic potential and transplanted autologous MSCs can be rapidly rejected after culture in FBS [32]. Therefore, new human‐derived alternatives have been evaluated as possible cell culture supplements to ensure that growth and expansion of MSCs are complain with current good manufacturing processes (GMPs).

Platelets are small enucleated cell fragments derived from megakaryocytes in the bone marrow and play a critical role in initiating hemostasis by binding and adhering to extracellular matrix components after endothelial injury, which in turns leads to platelet activation. Activated plate‐ lets then subsequently aggregate and become crosslinked with fibrin through activation of the coagulation cascade, thereby generating a platelet plug capable of blocking the flow of the blood. Platelets normally represent 0.1–0.25% of the blood and typically circulate for 5–9 days. Platelets are also an abundant source of growth factors, accounting for the majority of growth factors found in serum [33, 34]. As a result, various platelet extracts have been used for regen‐ erative medicine applications. Platelet lysate (PL) is one such supplement that has been utilized as a supplement for culture of MSCs and has been shown to be superior to FBS [35, 36]. We and several collaborators have performed extensive characterization of platelet lysate as a cell cul‐ ture supplement for expansion of human MSCs suitable for cell therapy trials. More recently, we have modified the PL production process such that PL may be used to form a 3D scaffold for MSC growth and invasion. In this chapter, we elaborate on our experience with PL as it pertains to culture of MSCs as well as describe a novel scaffold for cell delivery derived from PL extract.
