**Echinochrome A Promotes Ex Vivo Expansion of Peripheral Blood-Derived CD34**<sup>+</sup> **Cells, Potentially through Downregulation of ROS Production and Activation of the Src-Lyn-p110**δ **Pathway**

**Ga-Bin Park 1, Min-Jung Kim 1, Elena A. Vasileva 2, Natalia P. Mishchenko 2, Sergey A. Fedoreyev 2, Valentin A. Stonik 2, Jin Han 3, Ho Sup Lee 4, Daejin Kim 5,\* and Jee-Yeong Jeong 1,\***


Received: 21 August 2019; Accepted: 6 September 2019; Published: 9 September 2019

**Abstract:** Intracellular reactive oxygen species (ROS) play an important role in the proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). HSPCs are difficult to be expanded ex vivo while maintaining their stemness when they are exposed to oxidative damage after being released from the bone marrow. There have been efforts to overcome this limitation by using various cytokine cocktails and antioxidants. In this study, we investigated the effects of echinochrome A (Ech A)-a well-established and non-toxic antioxidant-on the ex vivo expansion of HSPCs by analyzing a CD34<sup>+</sup> cell population and their biological functions. We observed that Ech A-induced suppression of ROS generation and p38-MAPK/JNK phosphorylation causes increased expansion of CD34<sup>+</sup> cells. Moreover, p38-MAPK/JNK inhibitors SB203580 and SP600125 promoted ex vivo expansion of CD34<sup>+</sup> cells. We also demonstrated that the activation of Lyn kinase and p110δ is a novel mechanism for Ech A to enhance ex vivo expansion of CD34<sup>+</sup> cells. Ech A upregulated phospho-Src, phospho-Lyn, and p110δ expression. Furthermore, the Ech A-induced ex vivo expansion of CD34<sup>+</sup> cells was inhibited by pretreatment with the Src family inhibitor PP1 and p110δ inhibitor CAL-101; PP1 blocked p110δ upregulation and PI3K/Akt activation, whereas CAL-101 and PI3K/Akt pathway inhibitor LY294002 did not block Src/Lyn activation. These results suggest that Ech A initially induces Src/Lyn activation, upregulates p110δ expression, and finally activates the PI3K/Akt pathway. CD34<sup>+</sup> cells expanded in the presence of Ech A produced equal or more hematopoietic colony-forming cells than unexpanded CD34<sup>+</sup> cells. In conclusion, Ech A promotes the ex vivo expansion of CD34<sup>+</sup> cells through Src/Lyn-mediated p110δ expression, suppression of ROS generation, and p38-MAPK/JNK activation. Hence, Ech A is a potential candidate modality for the ex vivo, and possibly in vivo, expansion of CD34<sup>+</sup> cells.

**Keywords:** hematopoietic stem and progenitor cells; CD34<sup>+</sup> cells; ex vivo expansion; Lyn; Src; p110δ; ROS

#### **1. Introduction**

Hematopoietic stem and progenitor cell (HSPC) transplantation is widely used for the treatment of various hereditary diseases and blood-related malignancies, such as leukemia, and to promote hematologic recovery following anticancer therapy [1]. HSPCs can be harvested from bone marrow, umbilical cord blood (UCB), or mobilized peripheral blood (PB) using granulocyte colony-stimulating factor (G-CSF) administration for autologous and allogeneic transplantation [2]. Currently, PB-HSPC transplantation accounts for more than 60% of total HSPC transplantation worldwide, mainly due to the less invasive collection procedures [3]. However, the insufficient number of HSPCs, even after multiple days of collection, is a limiting factor for their clinical application of transplantation [4]. Several researchers are exploring ex vivo expansion of HSPCs to overcome this limitation; however, ex vivo expansion remains a difficult challenge for HSPC-based therapies.

Reactive oxygen species (ROS) are generated in the mitochondria; they regulate proliferation, differentiation, motility, and quiescence in many cell types, including HSPCs [5,6]. A previous study using a mouse model showed that increased levels of ROS can promote the differentiation of stem cells [7]. Quiescent HSPCs reside in a hypoxic niche in the bone marrow microenvironment that protects them from oxidative stress caused by excessive ROS production, mitochondrial dysfunction, or a combination of both [7,8]. HSPCs can proliferate and differentiate in the oxygen-rich vascular niche, resulting in increased intracellular ROS levels. ROS can regulate HSPC activity and various levels of ROS accumulation may affect the fate of HSPCs. High levels of ROS can trigger HSPC dysfunction, aging, and DNA damage. On the contrary, moderate levels of ROS are necessary for the proliferation, mobilization, and differentiation of HSPCs, and low-ROS cells have been shown to retain long-term self-renewal ability [7,9–11]. Culture media with appropriate cytokines are usually required for ex vivo cultures of HSPCs. However, it is still important to regulate the levels of ROS, as cytokine treatment itself triggers intracellular ROS generation [12]. Antioxidants can effectively remove excess ROS and maintain the redox balance of cells [13,14]. A recent study showed that N-acetyl cysteine (NAC) can reduce ROS levels to enhance ex vivo expansion of HSPCs [15].

Src family kinases (SFKs), including Lyn, Fyn, Fgr, Yes, Lck, Hck, Blk, and Trk, are well known for their contribution to malignant transformation and oncogenesis, and control downstream targets to regulate cell proliferation, differentiation, adhesion, migration, and the cell cycle [16]. Because of their role in cancer development and progression, SFKs have become critical targets for cancer therapy [17]. In the immune system, the most well-known function of SFKS is their role in integrin signaling [16]. Fyn and Lck kinases are also found in T cells and natural killer (NK) cells [18]. Lyn is a non-receptor tyrosine kinase that is predominantly found in the hematopoietic cells of myeloid and B lymphocyte lineages [19]. Lyn was originally identified as a hematopoietic-specific kinase; it is expressed in multiple tissues and is involved in the signaling of the B-cell receptor [20], GM-CSF receptor [21], erythropoietin (EPO) receptor [22], and c-kit [23]. Lyn phosphorylates several signaling molecules, including PI3K, FAK [24], ras-GAP, and Stat5 [25]. Lyn also plays an important role in acute myeloid leukemia (AML) cell proliferation [26], and the silencing of Lyn in imatinib-resistant chronic myelogenous leukemia (CML) cells can induce apoptosis [27]. Lyn activation induces p110 expression, whereas Lyn inhibition decreased migration in ovarian cancer cells exposed to cigarette smoke [28]. Colon cancer cells use Lyn for activation of the anti-apoptotic PI3K p110/Akt pathway and the induction of epithelial-mesenchymal transition (EMT) [29]. Therefore, several pieces of evidence show an important association of Lyn in both leukemia and solid tumor development. However, the exact roles and Lyn/Src activation in the relationship with ROS during the ex vivo expansion of HSPCs are still unclear.

Echinochrome A (Ech A) is a dark red pigment that is isolated from eggs, spines, and larvae of sea urchins [30]. Ech A is known to possess antioxidant, antiviral, antialgal, and antimicrobial activities [31,32]. Importantly, Ech A was shown to exhibit diverse intracellular antioxidant mechanisms, including the elimination of free radicals [33], inhibition of pulmonary fibrosis [34], and chelation of metal ions [35]. In this study, we demonstrate that Ech A is an effective agent to promote the

ex vivo expansion of G-CSF-mobilized PB-derived CD34<sup>+</sup> HSPCs through Src/Lyn-mediated p110δ upregulation, the suppression of ROS generation, and p38-MAPK/JNK activation.
