Manipulation of Hematopoietic Stem Cell Fate by Small Molecule Compounds

Abstract

Self-renewal and multipotential differentiation are two important features of hematopoietic stem/progenitor cells (HS/PCs) that make them as an ideal source of stem cells for treatment of many hematologic disorders and cancers. Regarding the limited number of cord blood HS/PCs, proper ex vivo expansion can significantly increase the clinical use of cord blood stem cells. Meanwhile, expansion of HS/PCs will be feasible through bypassing the quiescent state of HS/PCs and simultaneously enhancing their proliferative potential and survival while delaying the terminal differentiation and exhaustion. Previous investigations have demonstrated that defined sets of exogenous hematopoietic cytokines/growth factors such as stem cell factor, Flt-3 ligand, and thrombopoietin are able to expand HS/PCs. However, in recent years, small molecule compounds (SMCs) have emerged as a powerful tool for the effective expansion of HS/PCs by modulating multiple cellular processes including different signaling pathways and epigenetics. In this review, recent progress toward the use of SMCs in HS cell research is presented. We focus on the significant applications of SMCs related to HS/PC expansion and discuss the associated mechanism. At the end we present a list of those SMCs which enter to clinical trials.

Introduction

Hematopoietic stem/progenitor cells (HS/PCs) are defined by ability to long-term self-renewal and to generate all functional blood cells. Currently, transplantation of HS/PCs is widely used to treat various hematological disorders and malignancies [1 –3]. Umbilical cord blood (UCB) is one of the richest sources of HS/PCs. The limited number of cells in single UCB unit is sufficient to treat the child recipients, which also associate with delay in recovery of neutrophils and platelets. Ex vivo expansion of HS cells is a viable approach to overcome the drawbacks; however, this has not yet been fully achieved [4,5].

Comprehensive understanding about the regulators of HS/PC fate could help researchers to find the proper expansion protocol. An accumulating amount of studies have shown that various internal factors and external signals are involved in controlling the balance between self-renewal and differentiation processes of HSCs [6,7]. So far, several intrinsic factors have been identified, including transcription factors, signal transducers, antiapoptotic proteins, cell cycle regulators, and epigenetic modifiers, which play important roles in controlling the self-renewal and differentiation of HSCs (Fig. 1). Although the best expansion is achieved through manipulation of these intrinsic regulators, expansion with the help of extrinsic growth factors such as mesenchymal stromal cells and chemical modulators (cytokines and interleukins) might be a more preferred strategy in clinical applications [8,9]. Therefore, so far, most of the expansion protocols have used hematopoietic cytokines such as stem cell factor, thrombopoietin, Fms-like tyrosine kinase 3 ligand, interluekin-3, 6, 11, and granulocyte–macrophage colony-stimulating factor, but have not yet resulted in the expansion of high-quality clinical-grade cells.

FIG. 1.

Known regulators of HS/PC fate. The extrinsic factors (I, II, and III) dictated by niche regulate the activation of intrinsic factors (IV and V). After expression of downstream genes, hematopoietic stem cells can undergo several fates (VI). HS/PC, hematopoietic stem/progenitor cell. Color images available online at www.liebertpub.com/scd

In recent years, most of the researchers have not only used multiple combinations of the cytokines, but also small molecule compounds (SMCs), which are natural or synthetic low-molecular weight compounds (<900 Da). SMCs can easily penetrate cells and reversibly inhibit or promote function of various cell intrinsic factors. Furthermore, in comparison with the other regulatory molecules, SMCs are more stable and cost-effective and have less batch-to-batch activity variation. More importantly, SMCs minimize the controversial issues about the carcinogenesis, immune response, and ethical concerns that are often associated with the cells expanded ex vivo [10,11]. It should be noted that self-renewal is a complex process. On one hand, it requires active repression of differentiation, senescence, and apoptosis pathways, and on the other hand it promotes and/or maintaines the proliferation signals [12]. As shown in Fig. 2, small molecules can modulate all the mentioned processes through different molecular mechanisms.

FIG. 2.

Multiple uses of small molecules in regulation of HS/PC fate.

In this review, we introduce the SMCs that have been used in HS cell research with focus on their associated mechanisms. In addition, we present a summery of their clinical applications. We hope this review will be of interest to researchers and clinicians in the fields of HS cells.

Small Molecules Modulators of Cell Signaling Molecules

Kinase regulators

P38-MAPK inhibition

P38 mitogen-activated protein kinase (p38-MAPKs) is a member of mitogen-activated protein kinases super family that was identified originally as a stress-activated protein kinase. Although the kinase has a critical function in the production of erythroid [11,12] and myeloid cells [13], it is dispensable for self-renewal of HS/PCs [14]. Many studies have shown that upregulation of the cell cycle inhibitors such as p16 and p21 and finally HS/PC senescence during ex vivo expansion as well as under different physiological and pathological conditions are associated with elevated level of reactive oxygen species and p38 activation [15,16].

Zhou et al. found that a specific inhibitor of p38, SB203580 (Table 1), could dramatically expand either mouse bone marrow LSK (lineage-negative, Sca-1⁺, Kit⁺) cells [14] or hUCB-HS/PCs [17]. According to their results, SB significantly generates more CFU-GEMM (colony forming units–granulocytes, erythrocytes, macrophages, megakaryocytes), indicating that inhibition of p38 activity promotes expansion of more primitive HS/PCs. SB-treated cells also showed 30-fold greater engraftment potential in NOD/SCID mice than the cells cultured without SB. As SB-expanded cells are less positive for annexin-V and SA-β-gal (senescence-associated beta-galactosidase), promotion of expansion is likely attributable to SB-mediated inhibition of apoptosis/senescence processes that are correlated with downregulation of p16 and p21 mRNA.

Table 1.

Small Molecules as Modulators of Cell Signaling Molecules

	Small molecule	Mechanism/target	Key molecular effects	Cell system	Media/supplements	Culture period	References
Kinase regulators	SB203580	Inhibits p38 MAPK	↓ Senescence, apoptosis ↑ Expression of CXCR4 No significant effects on differentiation and proliferation	hUCB-CD133⁺ cells	Stemspan/SCF, TPO, Flt3 L	7 days	(2012) Ann. Hematology
	CHIR99021 + rapamycin	Inhibits GSK3 Inhibits mTOR pathway	↓ Protein synthesis Arrests cell cycle progression ↓ IL-2 signal transduction pathway	Mouse c-Kit⁺ cells	X-VIVO 15	7 days	(2012) Nature Medicine
	CHIR99021 + Insulin	Inhibits GSK3 Activates PI3K/Akt pathway	↓ Protein synthesis Arrests cell cycle progression ↓ IL-2 signal transduction pathway	Mouse LSK Flk^- cells	StemSpan/heparin, SCF, TPO	14 days	(2011) Genes & Development
	BIO	Inhibits GSK3β (activates WNT pathway)	↓ Expression of Notch and Tie2 signaling regulating genes ↑ Expression of CDK inhibitor p57 ↓ Expression of cyclin D1	hUCB-CD34⁺ cells	StemSpan/SCF, TPO, Flt3 L	4 days	(2011) Stem Cells
Regulators of transcription factors	SR1	Antagonizing aryl hydrocarbon receptor (AHR)	↑ Retention of CD34 expression ↑ Expansion of early multipotent progenitors	hUCB-CD34⁺ cells	Serum-free media/SCF, TPO, Flt3 L, IL6	7 days	(2010) Science
	DEAB	Inhibits aldehyde dehydrogenase	↓ Retinoic acid signaling pathway ↑ Expression of cEBP and HoxB4	Human BM and CB CD34⁺ CD38^-Lin^- cells	IMDM/10% FBS, SCF, TPO, Flt3 L,	7 days	(2006) Proc Nat l Acad Sci USA.
	OAC1	Induces OCT4 expression	↑ Expansion of early multipotent progenitors ↑ Expression of HOXB4 gene No significant effect on epigenome	hUCB-CD34⁺ cells	RPMI-1640/10% FBS, SCF, TPO, Flt3 L	4 days	(2015) Leukemia
Signal transducer activators	dm-PGE2	Activates prostaglandin receptors	↑ Homing, survival, and proliferation	hUCB-CD34⁺, CD133⁺ cells, MNC or HUVEC	IMDM/2% FCS	3, 6, or 9 h	(2011) Cell Stem Cell
Signal transducer activators	NR-101	Activates Tpo receptor (c-MPL)	→ c-MPL, STAT5 pathways ↑ VEGF expression Stabilizes HIF-1 protein	hUCB-CD34⁺ cells	Stemspan/SCF, TPO, Flt3 L	7 days	(2009) Experimental Hematology
Cell survival regulators	zVADfmk, zLLYfmk	Pan caspase inhibitor	↑ Expression of Bcl-2 ↓ Expression of caspase-3 → Notch1 ↑ Expansion of myeloid committed progenitors	hUCB-CD34⁺ cells	Stempro/SCF, TPO, Flt3 L, IL6	10 days	(2010) PLoS ONE
Cell cycle regulators	P18IN003, P18IN011	Inhibits p18^INK4C [cyclin-dependent kinase (inhibitors]	↑ Self-renewing division No effect on the cell proliferation rate	Mouse CD34 LKS^- cells	IMDM/10% FBS, SCF, TPO, Flt3 L	3 days	(2015) Nature Communications
Metabolic regulator of self-renewal	TEPA	Copper (Cu) chelator	↓ Activity of cytochrome c oxidase	hUCB-CD34⁺ cells	α-MEM, 10% FCS, SCF, TPO, Flt3 L, IL3, IL6	21 days	(2005) Experimental Hematology

Gray shading indicates clinically used small molecule, ↑ upregulation, ↓ downregulation, → activation.

GSK3 inhibition

Glycogen synthase kinase-3 is a kinase for >40 different substrates in a variety of intracellular signaling pathways involved in proliferation, migration, and apoptosis. GSK-3 also acts as a signaling hub that integrates several important modulators of hematopoiesis program such as Wnt, phosphatidylinositol 3-kinase (PI3K)/Akt, sonic hedgehog (SHh), and Notch signaling pathways [18]. As direct inhibition of GSK-3 activates the critical HSC self-renewal signaling pathways and enhances the hematopoietic repopulation, in recent years, many efforts have prompted to develop GSK-3 inhibitors as therapeutics.

One of the potent inhibitor of GSK3 is CHIR99021 (Table 1), which in combination with rapamycin, an mTOR inhibitor, significantly supports the expansion of functional long-term HSCs [19]. Moreover, the chir/rapamycin-treated cells show higher chimerism level in primary and secondary lethally irradiated mice after 4 months. Increased cells in the S/G2/M phase shows that chir/rapamycin promotes the HS/PC cycle progression while simultaneously inhibiting apoptosis and/or blocking differentiation. Chir in combination with insulin, a major stimulator of the PI3K/Akt pathway, can also drive self-renewal and expansion of long-term mouse LSK cells through Wnt activation [20].

It is hypothesized that GSK3 inhibitors exert their effects through the Wnt/β-catenin pathway, which has an important role in self-renewal and normal function of HS/PCs. Actually, Wnt activation blocks differentiation through downregulation of several differentiation-inducing transcription factors such as GATA and PU.1 as well as upregulation of Id2 proteins [21,22]. Although activation of Wnt pathway by chir alone leads to expansion of HS/PCs, in the presence of factors regulating the Wnt and PI3K or mTOR pathways, chir provides functional long-term HS/PCs through inducing proliferation and simultaneously inhibiting apoptosis and/or blocking differentiation (Fig. 3).

FIG. 3.

Mechanisms underlying the chemical manipulation of hematopoietic stem cell fate. Five small molecules and their targets within the pathways are shown. Green pointed arrows indicate activation and red blunt-end arrows repression. Color images available online at www.liebertpub.com/scd

A second GSK3 inhibitor is 6-bromoindirubin 3'-oxime (6-BIO; Table 1) that is as an inhibitor of hematopoietic differentiation. It has been shown that 5-day treatment of CD34⁺ cells with 0.5 μM results in the accumulation of late dividing cells and increased long-term proliferation of clonogenic progenitor/stem cells [23]. Upregulation of CDK inhibitor p57 and downregulation of cyclin-D1 are responsible for the delay that is seen in the cell cycle progression of 6-BIO-treated cells [24]. It should be noted that the frequency of SCID repopulating cell (SRC) is not increased in 6-BIO-expanded cell. However, only 24-h pretreatment with 6-BIO-can increase the regeneration potential of expanded cells, which leads to higher engraftment potential of CD34⁺ cells. Interestingly, the expression of Wnt/β-catenin target genes is not altered by 6-BIO, but rather the expression of several genes regulating Notch and Tie2 signaling is increased [24].

Regulators of transcription factors

Inhibition of retinoic acid receptors

Nuclear receptors (NRs) are ligand-activated transcription factors that are involved in diverse physiological functions such as metabolism, development, and reproduction [25 –28]. Therefore, these receptors are a rich source for the development of SMCs that mimic or block the action of endogenous ligands [29, 30].

The most characterized NRs that play important roles in hematopoiesis and HS/PC function are retinoic acid receptors (RARs and RXRs). In the nucleus, RA binds to and activates the receptors, leading to transcription of target genes that are involved in many important biological processes, including cell differentiation, proliferation, and lipid metabolism (Fig. 3). It has been found that the downstream RAR signaling in CD34⁺CD38⁻ cells is downregulated, whereas in more differentiated CD34⁺CD38⁺ cells it is activated [31]. So, it seems that RAR blockage maintains the primitive phenotype and function of HS/PCs. Chute et al. reported that inhibition of aldehyde dehydrogenase 1 (ALDH1)–an enzyme required for biosynthesis of retinoic acid from retinol–by using diethylaminobenzaldehyde (DEAB; Table 1) induces the expansion of human HS/PCs up to fourfold [32]. DEAB also caused a 3.4- and 7.7-fold expansion in the number of SRCs compared with the uncultured and control group, respectively. In addition, based on gene expression analysis, the expression of a positive regulator of HS/PC self-renewal, HOXB4 gene, is also upregulated in DEAB-expanded cells. In particular, DEAB-treated cells contain less number of colony forming cells, indicating that inhibition of RA signaling impeded in vitro maturation of HS/PCs.

Inhibition of aryl hydrocarbon receptor

Aryl hydrocarbon receptor is also a nuclear transcription factor that normally acts as a negative regulator of hematopoiesis through curbing excessive or unnecessary proliferation of HS/PCs [32]. Indeed, AhR expression is necessary for the proper maintenance of quiescence of HS/PCs and its downregulation allows HS/PCs to escape from quiescence, which subsequently leads to cell proliferation [33,34]. The footprint of AhR has been implicated in some hematopoiesis pathways such as β-catenin, CXCR4, and STAT5 [34,35]. StemRegenin 1 (SR1; Table 1) as an AhR antagonist was identified through screening a library of 100,000 heterocycles, which directly binds to AhR and inhibits its activity [35]. Culture of human-mobilized peripheral blood CD34⁺ cells in the presence of SR1 leads to 50- and 17-fold increase in number of CD34⁺ cells and SRCs, respectively. Moreover, SR1-expanded cells provide long-term engraftment potential in serial transplantation studies. Apparently, SR1 has no effect on division rate of CD34⁺ cells, but promotes the retention of CD34 expression and increases the number of multilineage CFUs through prevention of HS/PC differentiation. In phase 1/2 trial of SR1 (NCT01930162 and NCT01474681), transplantation of SR1-expanded CD34⁺ cells led to engraftment in 17 of 17 patients with significantly faster neutrophils and platelets recovery compared with the patients treated with unmanipulated UCB units [36].

Upregulation of OCT4

OCT4 (octamer-binding transcription factor 4) is a transcription factor of the POU family, which has a vital role in self-renewal of undifferentiated embryonic stem cells. Furthermore, the expression of OCT4 in a variety of adult stem cells suggests that it might have a role in somatic stem cells [37 –39]. The importance of OCT4 in hematopoietic fate transition was reported for the first time by Bhatia et al. They found that ectopic expression of OCT4 in human fibroblasts allowed the generation of human hematopoietic progenitor cells having the ability of CD45 expression and in vivo engraftment as well [40]. Broxmeyer and his colleagues, also, examined the role of OCT4-activating compound 1 (OAC1; Table 1) during ex vivo expansion of cord blood HS/PCs [41]. Culture of hUCB-CD34⁺ cells in the presence of an effective concentration of OAC1 (500 nM) for 4 days significantly increased the number of CD34⁺CD38⁻ cells (about 3.4-fold compared with uncultured group). The number of Lin^-CD34⁺CD38⁻CD45RA⁻CD90⁺CD49f⁺ cells increased to 7.6- and 2.8-fold, respectively, in comparison with uncultured and vehicle-treated group. OAC1 also increased cytokine-stimulated ex vivo expansion of HPCs, as there was significant increase in the number of GM, GEMM, and erythroid colonies. More importantly, the upregulation of SOX2 and NANOG along with OCT4 in OAC1-treated cells suggests that the well-known pluripotency regulatory complex of embryonic stem cells is likely to be involved in HS/PCs self-renewal network. The authors identified OCT4-HOXB4 axis as an essential mediator of OAC1 in HS/PC expansion.

Signal transducer activators

Agonist of prostaglandin receptors

Prostaglandin E2 (PGE2; Table 1) is the most abundant and most biologically active prostaglandin of mammalians, which its contribution in many diseases is associated with cell proliferation, apoptosis, angiogenesis, inflammation and immune surveillance [42,43]. It has also been shown that PGE2 has a regulatory role in hematopoiesis through inhibiting myelopoiesis while promoting erythroid and multilineage colony formation [44]. Only 2-h ex vivo exposure to a long-acting prostaglandin E2 analogue (dmPGE2) can make a twofold increase in HS/PC cycle activity and a twofold increase in HS/PC homing, which totally leads to fourfold higher SRC frequency [45]. It is well known that bone marrow-secreted chemoattractants stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 have an important role in homing and proper engraftment of HS/PCs [46]. The effect mediated by PGE2 was thought to be associated with increased expression of CXCR4 with subsequent chemotactic response to SDF-1 (Fig. 3). PGE2 can also reduce the activation of intracellular active caspase-3 and promote the expression of survivin, a member of the inhibitor of apoptosis protein family [47].

In a clinical trial (NCT00890500), incubation with PGE2 was investigated on adults with hematologic malignancies [48]. Incubation the UCB with dmPGE2 before infusion leads to faster neutrophil recovery along with long-term engraftment in 10 of 12 treated participants.

Agonist of TPO receptor

Thrombopoietin (TPO) is a cytokine that is mainly responsible for megakaryocyte differentiation, but its role in HSC survival and maintenance during adult hematopoiesis is also noteworthy [49,50]. Previous studies have shown that TPO can activate three major pathways: JAK/STAT (Janus kinase/signal transducer and activator of transcription) [51], Ras/MAPK (mitogen-activated protein kinase) [52], and PI3K/AKT [53]. Moreover, TPO can also activate the HIF signaling cascade that is an essential pathway for the maintenance of HSCs [54]. The function of TPO is linked to a cell surface receptor named myeloproliferative leukemia protein (c-MPL), which is expressed in the megakaryocytes, HSCs, and HPCs. It has been shown that TPO can markedly augment the ex vivo expansion of human cord blood-derived hematopoietic progenitors in combination with stem cell factor and flt3 ligand [55].

A novel c-MPL agonist, NR-101 (Table 1), has been identified, which could efficiently increase the number of CD34⁺ or CD34⁺CD38⁻ cells with a greater than twofold increase compared with TPO [56]. Interestingly, although Tpo-treated cells showed no significant change in number of SRCs, NR-101 can increase the number of SRCs 2.3-fold compared with Tpo-treated cells. Consistently, the expression of HIF target genes such as vascular endothelial growth factor and also the genes involved in glycolysis and glucose transport (which might favor the maintenance and/or expansion of HS/PCs) was enhanced more efficiently in NR-101-treated cells. In summary, it seems that NR-101 could be a suitable substitute for Tpo to achieve a more efficient ex vivo expansion of HS/PCs.

Cell cycle regulators

Targeting cell cycle progression has not been a successful strategy for HS/PC expansion, because stimulating the cell division is often associated with cellular exhaustion [57,58]. However, cyclin-dependent kinase inhibitors (CKIs), which negatively control the cell cycle, are a good target for stem cell regulation as they indirectly regulate the cell proliferation. The G1 phase of the mammalian cell cycle is a critical interface in which the somatic stem cell fate may be determined [59,60]. The members of INK4 family (p16^INK4a, p15^INK4b, p18^INK4c, and p19^INK4d) are a group of CKIs that block progression of the cell cycle G1 checkpoint by inhibiting CDK4/6 (Fig. 4). It has been shown that deletion of the early G1-phase inhibitor, p18^INK4C, results in improved long-term engraftment, largely by increasing self-renewing divisions of the primitive HS/PCs [61,62]. Using mouse model and single-cell analysis, it has been indicated that p18 is a potent inhibitor of HS/PC self-renewal, whereby increased self-renewing division of HS/PC is seen in its absence [61]. They identified two p18 small molecular inhibitors, P18IN003 and P18IN01 (Table 1), which can specifically block the activity of p18 protein. The authors also showed that the compounds could significantly expand more primitive hematopoietic cells during ex vivo culture, which have higher long-term repopulating ability than the control group.

FIG. 4.

Regulation of HSC cell cycle by small molecules. The small molecules promote the entrance of quiescent HSCs into G1 phase through an inhibitory mechanism that regulates the activity of CyclinD–CDK4/6 complexes. A, acetyl group; M, methyl group. Color images available online at www.liebertpub.com/scd

Cell survival regulators

Cell exhaustion and apoptosis are major reasons of short-lived and nonfunctional HS/PCs in culture. Apoptosis is a major factor that determines the size of hematopoietic cell mass in both normal and pathological conditions [63 –65]. So, many researchers tried to find novel small molecules with prosurvival activities, which are discussed in the next paragraph.

Caspase and calpain are two cysteine proteases that their roles have been highly implicated in apoptotic cell death and necrosis as well [66]. A novel role of apoptotic protease inhibitors was observed by Sangeetha et al. [67]. They found that in the presence of a combination of caspase/calpain inhibitors, zVADfmk/zLLYfmk (Table 1), the promotion of hUCB-HS/PC expansion was accompanied by a significant reduction in the Annexin-V⁺ population, increase in the bcl-2⁺ population, and significant reduction in the expression of major members of the apoptotic machinery such as caspase 1, 3, 8, and Fas antigen. These results were confirmed by higher clonogenicity and long-term culture initiating potential of the cells expanded in the presence of zVADfmk/zLLYfmk. NOD/SCID mouse transplantation assay showed that zVADfmk/zLLYfmk-treated cells support higher long-term engraftment and an efficient regeneration of major lymphomyeloid lineages in the bone marrow of hosts compared with the positive control cell recipients. In the next study, they found that in the zVADfmk/zLLYfmk-treated cells, the CXCR4 protein, integrins, and adhesion molecules are also upregulated, which results in a higher migration and adhesive interactions in vitro and a significantly enhanced homing to the bone marrow of NOD/SCID mice [68].

Metabolic regulator of self-renewal

Recent advances in metabolic analysis of stem cells have demonstrated that metabolic processes may contribute to manage the decision between self-renewal and differentiation [69,70]. For instance, there are cumulative studies indicating that remaining in a quiescent state required self-renewing HS/PCs to live in a hypoxic niche [71 –73]. Thus, to support ATP production, HS/PCs must rely heavily on anaerobic glycolysis, rather than mitochondrial oxidative phosphorylation [74,75].

Cu is the cofactor of cytochrome c oxidase, which is the key enzyme in mitochondrial respiratory chain and oxidative phosphorylation system. As shown in Fig. 5, tetraethylenepentamine (TEPA; Table 1) by reducing the cellular Cu content attenuates the activity of cytochrome c oxidase, which, in turn, leads to a switch in metabolic pathways toward anaerobic glycolysis [76]. The critical role of Cu in modulating hematopoiesis is supported by the finding that Cu deficiency leads to shortage of mature functional circulating blood cells [77]. Peled et al. reported that TEPA could decrease the Cu pool of the cells and then promote the preferential proliferation of human CD34⁺CD38⁻ cells compared with a control group, which results in an enhanced reconstitution capacity of these cells in NOD-SCID. The feasibility and safety of transplantation of CD133⁺ cord blood hematopoietic progenitors cultured for 3 weeks in the presence of TEPA have been shown. At present, a global pivotal phase 2/3 registration study is underway to evaluate the safety and efficacy of 21-day StemEx-expanded CB unit in patients with advanced hematological malignancies [78].

FIG. 5.

TEPA modulates the proliferation of hematopoietic stem cells through reducing the cellular Cu content and attenuating the activity of cytochrome c oxidase, which, in turn, leads to a switch in metabolic pathways toward anaerobic glycolysis. TEPA, tetraethylenepentamine. Color images available online at www.liebertpub.com/scd

Epigenetic Modulation by Small Molecules

The epigenetics refers to reversible remodeling of chromatin that is tightly regulated by two major mechanisms: DNA methylation induced by DNA methyltransferases (DNMTs) and histone modifications induced by histone deacetylases (HDACs) and histone acetyltransferases (HATs). As shown in Fig. 6, epigenetic mechanisms can control the gene expression through chromatin remodeling and accessibility of regulatory transcription proteins to the condensed genomic DNA. There is some evidence that epigenetic mechanisms have an important role in modulating the division rate of HS/PCs and balancing the symmetrical and asymmetrical divisions [79 –81]. Thus, modifying the epigenetic processes with the help of small molecule drugs alone or in combination with each other is increasingly being used to enhance expansion efficacy of HS/PCs, which are summarized in the next paragraphs.

FIG. 6.

Modulation of hematopoietic stem cell fate by regulation of epigenetic mechanisms. Epigenetic modulators can modify chromatin structure and make it more permissive to transcriptional machinery. Color images available online at www.liebertpub.com/scd

Inhibition/activation of NAD-dependent HDAC (SIRT1)

Sirtuin1, a mammalian NAD-dependent deacetylase, catalyzes the removal of an acetyl group from histones and nonhistone proteins including transcription factors. Accumulative studies have indicated that SIRT1 can regulate various cellular processes, including metabolism [82], differentiation [83], cell survival [84], cellular senescence [85], inflammation–immune function [82,86], and is also involved in several human disorders such as obesity, cancer, and aging. Therefore, it is predicted that modulators of SIRT1 could be raised as new therapeutic tools. For example, nicotinamide (NAM; Table 2) is one of the noncompetitive inhibitors of SIRT1, which can regulate granulocytic differentiation of promyelocytic leukemia cell line [87]. Low concentration of NAM can also attenuate ex vivo differentiation and promote long-term expansion of cultured CD34⁺ cells [88]. Interestingly, NAM-expanded cells showed higher efficacy in bone marrow homing, whereas there was no change in CXCR4 gene expression. The data suggest that superior homing and engraftment of NAM-treated cells are associated with the modulation of CXCR4 downstream signaling pathways [88]. NAM has been tested in a phase 1 clinical trial (NCT01816230). Based on the gained results, transplantation of NAM-expanded cord blood cells is associated with faster neutrophil engraftment, fewer total and bacterial infections, and shorter hospitalization in the first 100 days than standard UCB transplantation.

Table 2.

Small Molecules as Epigenetic Modulators

	Small molecule	Mechanism/target	Key molecular effects	Cell system	Media/supplements	Culture period	References
Modulation of NAD-dependent HDAC	Nicotinamide	Inhibits NAD-dependent histone/protein deacetylase sirtuin-1 (SIRT1)	↓P21 expression	hUCB-CD34⁺ cells	α-MEM/10% FBS, SCF, TPO, Flt3 L, IL6	21 days	(2012) Experimental Hematology
Modulation of NAD-dependent HDAC	Resveratrol	Activates NAD-dependent histone/protein deacetylase sirtuin-1 (SIRT1)	↑ Expansion of CD34⁺/CD133⁺ ↑ Expression of cell cycle–associated genes ↓ Cyclooxygenase 1	hUCB-CD34⁺ cells	Stemspan/SCF, TPO, Flt3 L, IL6	9 days	(2015) Stem Cells Translational Medicine
Inhibition of HATs	Garcinol	Inhibits histone acetyltransferases (HATs)	↑ Expression of AMICA1, BTG2, HLF ↓ Expression of IL8, PF4, and PPBP	hUCB-CD34⁺ cells	Stemspan/SCF, TPO, Flt3 L	7 days	(2011) Plos ONE
Inhibition of DNMTs/HDACs	5-AzaD +TSA	Inhibits DNA methyltransferase (DNA MTase) Inhibits histone deacetylase classes I and II	↑ Expression of HOXB4, Bmi-1, GATA-2, GATA-1, Notch1 ↑ Expression of cell cycle regulator genes p21, p27	hUCB-CD34⁺ cell	IMDM/30% FBS, SCF, TPO, Flt3 L, IL3, MGDF	7 days	(2006) Blood
	VPA	Inhibits histone deacetylase (HDAC1)	↑ Histone H4 acetylation levels at AC133 and HOXB4 regulatory sites↑ Expression of KDR, AC133, c-kitR, GATA1, and HOXB4	Human BM, PB, and CB-CD34⁺ cells	IMDM/10% human AB serum, SCF, TPO, Flt3 L, IL3	7 days	(2005)Cancer Res

Gray shading indicates clinically used small molecule, ↑ upregulation, ↓ downregulation, → activation.

Unlike NAM, resveratrol (Rvt, trans-3,5,4′-trihydroxystilbene; Table 2) is a natural polyphenol compound that activates SIRT1. Accumulative evidence has shown that Rvt can affect various cellular processes such as cellular metabolism [89], nuclear factor κB (NF-κB) [90], PI3K/Akt/mTOR signaling pathways [91], and inhibition of vascular cell adhesion molecules [91] and cyclooxygenases [92]. So, it seems that Rvt could be used to treat diseases affected by abnormal metabolic control, inflammation, and cell cycle defects. In the field of HS cell research, it has been found that UCB-CD34⁺ cells that cultured in the basic cytokine medium (contained SCF, Tpo, Flt3 L, and IL-6) in the presence of Rvt (10 μM) were expanded to 27-fold ±9 and 3.5-fold ±0.9 in the number of total and CD34⁺CD133⁺ cells, respectively [93]. Moreover, Rvt-expanded cells had a higher engraftment level not only in the bone marrow of NSG mice but also in the peripheral blood of recipients. Rvt-cultivated cells also supported a robust multilineage engraftment in primary and secondary recipients. Based on gene set enrichment analysis, the expansion effect mediated by Rvt was thought to be associated with enhanced cell cycle-associated genes on the one side and preserved characteristics of HS/PCs on the other side. Although there are many clinical data available regarding the pharmacological action of resveratrol on cancer, neurological disorders, cardiovascular diseases, diabetes, nonalcoholic fatty liver disease, and obesity, yet it has not been used in blood disease clinical trials [94].

Inhibition of HATs

HATs that catalyze the acetylation of histone proteins in reverse of HDACs are also key players in epigenetic regulation of HS cell fate [95,96]. Nishino et al. screened a library of 92 biologically active natural compounds and identified Garcinol (GAR; Table 2)–a nonspecific inhibitor of HATs–and its derivative Iso-garcinol as effective stimulators for ex vivo expansion of CD34⁺CD38^- cells [97]. They reported that CFU-GEMM, which represents the most primitive progenitors, is more frequently contained in the presence of GAR and ISO. The frequency of SRCs is also increased up to 2.2-fold by applying GAR. Therefore, the enhanced engraftment potential of GAR-treated CD34⁺ cells is attributed to increased number of SRCs rather than augmented homing capacity of the cells. Unexpectedly, in the presence of GAR, there is no change in the expression of some important self-renewal signaling pathways such as HOXB4 and Notch1. Nevertheless, the hepatic leukemia factor (HLF) gene (which protects HS/PCs against apoptosis and enhances their in vivo reconstitution capacity) is upregulated by GAR.

Inhibition of DNMTs/HDACs

Araki et al. attempted to reverse the silencing status of the expanded HS/PCs by combination of two epigenetic processes: DNA demethylation and histone hyperacetylation [98]. They found that sequential treatment of human cord blood CD34⁺ cells with a DNMT inhibitor, 5-aza-2'-deoxycytidine (5azaD; Table 2), followed by a HDAC inhibitor, Trichostatin A (TSA; Table 2), leads to significantly increase in expansion ability, clonogenic potential, cell division rate, and engraftment potential of CD34⁺CD90⁺ cells [98]. The gene expression analysis of 5azaD/TSA-treated cells implicated the changes in Notch1, HOXB4, BMI1, and GATA2 transcription factors, which are important in self-renewal of HS/PCs. Moreover, p21 and p27 of cell cycle inhibitor genes were upregulated, whereas the cell proliferation-related gene C-MYC was downregulated. In the next step, they assessed the efficacy of several HDAC inhibitors including valproic acid (VPA; Table 2) alone or in combination with 5azaD [99]. It was found that VPA has the best effect on the expansion of CD34⁺CD90⁺ and progenitor cells; however, VPA treatment only permits the maintenance of HS/PCs that lack serial transplantation ability. By contrast, another study has reported that VPA-expanded cells have multilineage engraftment in primary and secondary NSG mice [100]. Further studies about the effectiveness of VPA on the maintenance of hematopoietic potential of cultured cells revealed that in the presence of VPA, CD34⁺ cells have lower proliferation rate and longer G0/G1 phase. This fact is consistent with the high preservation of CD34⁺ cells after 7 days ex vivo expansion in the presence of VPA. The expansion effect mediated by VPA was thought to be associated with an increasing level of histone H4 at specific regulatory sites on HOXB4, a transcription factor gene with a key role in the regulation of HS/PC self-renewal and AC133, a recognized marker for HS/PCs. A global microarray analysis revealed that CD34⁺ cells expanded in 5azaD/TSA and VPA presumably represent the expansion and maintenance of in vivo repopulating HS/PCs, respectively [99].

Small Molecules with Unknown Mechanism/Target

In recent years, several small molecules have been discovered. Although their effects on the ex vivo expansion of HS/PCs have been indicated and numerous downstream targets were also identified, the exact molecular mechanisms of these small molecules have not yet been fully understood. Thus, further investigation of the mechanism for these compounds may lead to the discovery of new regulators of HS/PCs self-renewal.

UM171

Fares et al. tested a library comprising >5000 low molecular weight (LMW) molecules in a phenotypical screen based on ex vivo expansion of CD34⁺CD45RA^--mobilized peripheral blood cells. Using an optimized fed-batch culture system, they found that a pyrimido-[4, 5-b]-indole derivative, UM171 (Table 3), can enhance the ex vivo self-renewal of human HS/PCs [101]. UM171 has no effect on the division rate of cultured cells, whereas it promotes the retention of CD34⁺CD45RA^- phenotype. It also reduces transcripts associated with erythroid and megakaryocytic differentiation. Interestingly, the most highly upregulated gene in the UM171-treated cells was PROCR (also called EPCR or CD201), which recently has been discovered as a novel marker for mouse LT-HS/PCs [102,103]. UM171 has also a great potential in derivation of hematopoietic progenitor cells from human pluripotent stem cells [104].

Table 3.

Small Molecules with Unknown Mechanism/Target

Small molecule	Mechanism/target	Key molecular effects	Cell system	Media/ supplements	Culture period	References
UM171	Unknown	↑ Expansion of progenitor cells↑ Expression of PROCR (EPCR or CD201) gene↓ Differentiation	Human PB CD34⁺ CD45RA⁻ cells	Stemspan/SCF, TPO, Flt3L, low-densitylipoproteins	12 days	(2014)Science
Serotonin (5-HT)	Unknown	→ 5-HT receptor↓ Caspase-3 expression	hUCB-CD34⁺ cells	QBSF-60/ SCF, TPO, Flt3L, IL6	8 days	(2007)Stem Cells

Gray shading indicates clinically used small molecule, ↑ upregulation, ↓ downregulation, → activation.

According to the primary results gained from phase 1/2 trial of UM171 (NCT02668315), transplantation of 7-day UM171-expanded CB unit appears feasible and provides clinical benefit beyond faster engraftment with fewer infectious complications and better HLA matching [105].

5-Hydroxtryptamine

5-Hydroxtryptamine (5-HT, serotonin; Table 3) is a neurotransmitter that not only has a critical role in the central nervous system but also has proproliferative and antiapoptotic effect through Ras or MAPK pathways [106 –108]. There is also some evidence that 5-HT has essential function in embryogenesis [109,110]. In 1996, Yang et al. reported that 5-HT can stimulate megakaryocytopoiesis through 5-HT receptors (5-HTR) [111]. Later, they demonstrated that in the presence of serotonin, the expansion of either total nuclear cells or CD34⁺CD38⁻ cells, the number of CFU fibroblasts, and also the engraftment potential of 5-HT-treated CD34⁺ cells were increased [112]. They found that serotonin can also significantly reduce the number of cells that are in early or late phase of apoptosis. The antiapoptotic effect of serotonin is thought to be mediated through mitochondria signaling, reduced caspase activation, and Akt/ERK1/2 pathways [113,114].

Conclusion and Perspective

Although much effort has been made toward the HS/PC transplantation therapy, production of desirable HS/PCs that can be used for cell therapy is still in early developmental stages. With the growing knowledge about the mechanisms involved in proliferation, differentiation, and homing of HS/PCs, effective approaches that exert precise control of cellular signaling pathways have emerged as a tool for successful ex vivo expansion of the cells. Chemical approaches not only represent a powerful tool for maintaining the HS/PC self-renewal, but also provide the means for studying the involved mechanisms. More importantly, chemical molecules are also finding their ways to the clinic as potential therapeutic agents.

Two different approaches have been used to find effective SMCs. One way is screening libraries to discover unknown regulators or new signaling pathways that govern proliferation, self-renewal, and maintenance of HS/PCs. Another way is hypothesis-based selection of small molecules that target the known signaling pathways. Till now, although many useful small molecules have been identified that alone or in combination with other small molecules can promote the HS/PC self-renewal, none of them compensates the need for three major hematopoietic cytokines, including SCF, Tpo, and Flt3 L except MPL. Therefore, further studies should be continued for identification of chemical molecules that can be used as an alternative for cytokines.

Moreover, it can be seen that increase in fold expansion and engraftment ability varies among the studied small molecules. For example, although most of the used SMCs can increase fold expansion of CD34⁺ cord blood cells up to 2-5-fold, the SR1 small molecule can expand the cells by ∼50-fold. It is worthy to note that these small molecules were tested in different cell phenotypes and under different culture conditions, which could make variation in functions/effects of the small molecules. Therefore, to identify the best SMCs, an identical and standard culture protocol would be invaluable. More importantly, whether the cells expanded by such small molecules maintain their in vivo physiological function remains to be fully determined. We hope that the ongoing discovery of new SMCs will continue to ultimate ex vivo generation of correct cells that eventually can be used in clinics.

Footnotes

Acknowledgments

The authors thank members of the hematopoietic stem cell group of Royan Institute for their discussions and critiques of this article. We apologize to all scientists whose research could not be discussed and cited in this review owing to space limitations. Dr. M. Ebrahimi is supported by Royan Institute (Code: 91000597), and also supported partly by the Royan Stem Cell Technology Company.

Author Disclosure Statement

The authors declare no potential conflicts of interest with respect to the research, authorship, or publication of this article.

References

Mahmoud

, Elhaddad

, Fahmy

, Samra

, Abdelfattah

, El-Nahass

, Fathy

and Abdelhady

. (2015). Allogeneic hematopoietic stem cell transplantation for non-malignant hematological disorders. J Adv Res, 6:449–458.

Wildes

, Stirewalt

, Medeiros

and Hurria

. (2014). Hematopoietic stem cell transplantation for hematologic malignancies in older adults: geriatric principles in the transplant clinic. J Natl Compr Canc Netw, 12:128–136.

Shahrokhi

, Menaa

, Alimoghaddam

, McGuckin

and Ebtekar

. (2012). Insights and hopes in umbilical cord blood stem cell transplantations. J Biomed Biotechnol, 2012:572821.

Park

, Yoo

and Kim

. (2015). Hematopoietic stem cell expansion and generation: the ways to make a breakthrough. Blood Res, 50:194–203.

Sotnezova

, Andreeva

, Grigoriev

and Buravkova

. (2016). Ex vivo expansion of hematopoietic stem and progenitor cells from umbilical cord blood. Acta Nat, 8:6–16.

, Nordon

, O'Brien

, Symonds

and Dolnikov

. (2017). Ex vivo expansion of hematopoietic stem cells to improve engraftment in stem cell transplantation. Methods Mol Biol, 1524:301–311.

Wang

and Ema

. (2016). Mechanisms of self-renewal in hematopoietic stem cells. Int J Hematol, 103:498–509.

and Cheng

. (2016). Chemical transdifferentiation: closer to regenerative medicine. Front Med, 10:152–165.

Zhang

, Li

, Laurent

and Ding

. (2012). Small molecules, big roles–the chemical manipulation of stem cell fate and somatic cell reprogramming. J Cell Sci, 125:5609–5620.

10.

Lin

, Zhao

, Xiao

and Li

. (2015). Mechanisms determining the fate of hematopoietic stem cells. Stem Cell Investig, 2:10.

11.

Uddin

, Ah-Kang

, Ulaszek

, Mahmud

and Wickrema

. (2004). Differentiation stage-specific activation of p38 mitogen-activated protein kinase isoforms in primary human erythroid cells. Proc Natl Acad Sci U S A, 101:147–152.

12.

Dalmas

, Tierney

, Zhang

, Narayanan

, Boyce

, Schwartz

, Frazier

and Scicchitano

. (2008). Effects of p38 MAP kinase inhibitors on the differentiation and maturation of erythroid progenitors. Toxicol Pathol, 36:958–971.

13.

Geest

, Buitenhuis

, Laarhoven

, Bierings

, Bruin

, Vellenga

and Coffer

. (2009). p38 MAP kinase inhibits neutrophil development through phosphorylation of C/EBPalpha on serine 21. Stem Cells, 27:2271–2282.

14.

Ito

, Hirao

, Arai

, Matsuoka

, Takubo

, Ikeda

and Suda

. (2005). Inactivation of p38 MAPK extends self-renewal capacity of haematopoietic stem cells. Blood, 106:265–273.

15.

Ito

, Hirao

, Arai

, Takubo

, Matsuoka

, Miyamoto

, Ohmura

, Naka

, Hosokawa

, Ikeda

and Suda

. (2006). Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells. Nat Med, 12:446–451.

16.

Hosokawa

, Arai

, Yoshihara

, Nakamura

, Gomei

, Iwasaki

, Miyamoto

, Shima

, Ito

and Suda

. (2007). Function of oxidative stress in the regulation of hematopoietic stem cell-niche interaction. Biochem Biophys Res Commun, 363:578–583.

17.

Zou

, Zou

, Wang

, Li

, Wang

, Zhou

and Liu

. (2012). Inhibition of p38 MAPK activity promotes ex vivo expansion of human cord blood hematopoietic stem cells. Ann Hematol, 91:813–823.

18.

McCubrey

, Rakus

, Gizak

, Steelman

, Abrams

, Lertpiriyapong

, Fitzgerald

, Yang

, Montalto

, et al. (2016). Effects of mutations in Wnt/beta-catenin, hedgehog, Notch and PI3K pathways on GSK-3 activity-Diverse effects on cell growth, metabolism and cancer. Biochim Biophys Acta, 1863:2942–2976.

19.

Huang

, Nguyen-McCarty

, Hexner

, Danet-Desnoyers

and Klein

. (2012). Maintenance of hematopoietic stem cells through regulation of Wnt and mTOR pathways. Nat Med, 18:1778–1785.

20.

Perry

, He

, Sugimura

, Grindley

, Haug

, Ding

and Li

. (2011). Cooperation between both Wnt/β-catenin and PTEN/PI3K/Akt signaling promotes primitive hematopoietic stem cell self-renewal and expansion. Genes Dev, 25:1928–1942.

21.

Ruiz-Herguido

, Guiu

, D'Altri

, Inglés-Esteve

, Dzierzak

, Espinosa

and Bigas

. (2012). Hematopoietic stem cell development requires transient Wnt/β-catenin activity. J Exp Med, 209:1457–1468.

22.

Sturgeon

, Ditadi

, Awong

, Kennedy

and Keller

. (2014). Wnt signaling controls the specification of definitive and primitive hematopoiesis from human pluripotent stem cells. Nat Biotechnol, 32:554–561.

23.

Trowbridge

, Xenocostas

, Moon

and Bhatia

. (2006). Glycogen synthase kinase-3 is an in vivo regulator of hematopoietic stem cell repopulation. Nat Med, 12:89–98.

24.

, Holmes

, Palladinetti

, Song

, Nordon

, O'Brien

and Dolnikov

. (2011). GSK-3beta inhibition promotes engraftment of ex vivo-expanded hematopoietic stem cells and modulates gene expression. Stem Cells, 29:108–118.

25.

Moschetta

. (2015). Nuclear receptors and cholesterol metabolism in the intestine. Atheroscler Suppl, 17:9–11.

26.

Skerrett

, Malm

and Landreth

. (2014). Nuclear receptors in neurodegenerative diseases. Neurobiol Dis, 72(Pt A):104–116.

27.

Polvani

, Tarocchi

, Tempesti

and Galli

. (2014). Nuclear receptors and pathogenesis of pancreatic cancer. World J Gastroenterol, 20:12062–12081.

28.

Gonzalez-Sanchez

, Firrincieli

, Housset

and Chignard

. (2015). Nuclear receptors in acute and chronic cholestasis. Dig Dis, 33:357–366.

29.

Malek

. (2014). Nuclear receptors as potential therapeutic targets for age-related macular degeneration. Adv Exp Med Biol, 801:317–321.

30.

Burris

, Solt

, Wang

, Crumbley

, Banerjee

, Griffett

, Lundasen

, Hughes

and Kojetin

. (2013). Nuclear receptors and their selective pharmacologic modulators. Pharmacol Rev, 65:710–778.

31.

Ghiaur

, Yegnasubramanian

, Perkins

, Gucwa

, Gerber

and Jones

. (2013). Regulation of human hematopoietic stem cell self-renewal by the microenvironment's control of retinoic acid signaling. Proc Natl Acad Sci U S A, 110:16121–16126.

32.

Chute

, Muramoto

, Whitesides

, Colvin

, Safi

, Chao

and McDonnell

. (2006). Inhibition of aldehyde dehydrogenase and retinoid signaling induces the expansion of human hematopoietic stem cells. Proc Natl Acad Sci U S A, 103:11707–11712.

33.

Lindsey

and Papoutsakis.

(2012). The evolving role of the aryl hydrocarbon receptor (AHR) in the normophysiology of hematopoiesis. Stem Cell Rev, 8:1223–1235.

34.

Smith

, Rozelle

, Leung

, Ubellacker

, Parks

, Nah

, French

, Gadue

, Monti

, et al. (2013). The aryl hydrocarbon receptor directs hematopoietic progenitor cell expansion and differentiation. Blood, 122:376–385.

35.

Boitano

, Wang

, Romeo

, Bouchez

, Parker

, Sutton

, Walker

, Flaveny

, Perdew

, et al. (2010). Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells. Science, 329:1345–1348.

36.

Wagner

Jr. , Brunstein

, Boitano

, DeFor

, McKenna

, Sumstad

, Blazar

, Tolar

, Le

, et al. (2016). Phase I/II Trial of StemRegenin-1 expanded umbilical cord blood hematopoietic stem cells supports testing as a stand-alone graft. Cell Stem Cell, 18:144–155.

37.

Galiger

, Kostin

, Golec

, Ahlbrecht

, Becker

, Gherghiceanu

, Popescu

, Morty

, Seeger

and Voswinckel

. (2014). Phenotypical and ultrastructural features of Oct4-positive cells in the adult mouse lung. J Cell Mol Med, 18:1321–1333.

38.

Wei

and Shen

. (2011). Transcriptional regulation of oct4 in human bone marrow mesenchymal stem cells. Stem Cells Dev, 20:441–449.

39.

Ono

, Kajitani

, Uchida

, Arase

, Oda

, Nishikawa-Uchida

, Masuda

, Nagashima

, Yoshimura

and Maruyama

. (2010). OCT4 expression in human uterine myometrial stem/progenitor cells. Hum Reprod, 25:2059–2067.

40.

Szabo

, Rampalli

, Risueño

, Schnerch

, Mitchell

, Fiebig-Comyn

, Levadoux-Martin

and Bhatia

. (2010). Direct conversion of human fibroblasts to multilineage blood progenitors. Nature, 468:521–526.

41.

Huang

, Lee

, Cooper

, Hangoc

, Hong

, Chung

and Broxmeyer

. (2016). Activation of OCT4 enhances ex vivo expansion of human cord blood hematopoietic stem and progenitor cells by regulating HOXB4 expression. Leukemia, 30:144–153.

42.

Kozak

, Milne

, Bentzen

and Yock

. (2012). Elevation of prostaglandin E2 in lung cancer patients with digital clubbing. J Thorac Oncol, 7:1877–1878.

43.

Suzuki

, Ogawa

, Watanabe

, Takayama

, Hirata

, Nagai

and Isobe

. (2011). Roles of prostaglandin E2 in cardiovascular diseases. Int Heart J, 52:266–269.

44.

Pelus

and Hoggatt

. (2011). Pleiotropic effects of Prostaglandin E(2) in hematopoiesis; Prostaglandin E(2) and other eicosanoids regulate hematopoietic stem and progenitor cell function. Prostaglandins Other Lipid Mediat, 96:3–9.

45.

Pelus

, Hoggatt

and Singh

. (2011). Pulse exposure of haematopoietic grafts to prostaglandin E2 in vitro facilitates engraftment and recovery. Cell Prolif, 44(Suppl 1):22–29.

46.

Hoggatt

, Singh

, Sampath

and Pelus

. (2009). Prostaglandin E2 enhances hematopoietic stem cell homing, survival, and proliferation. Blood, 113:5444–5455.

47.

Bai

, Jiang

, Ding

, Peng

, Ma

, Wang

, Zhang

and Leng

. (2010). Prostaglandin E2 upregulates survivin expression via the EP1 receptor in hepatocellular carcinoma cells. Life Sci, 86:214–223.

48.

Cutler

, Multani

, Robbins

, Kim

, Le

, Hoggatt

, Pelus

, Desponts

, Chen

, et al. (2013). Prostaglandin-modulated umbilical cord blood hematopoietic stem cell transplantation. Blood, 122:3074–3081.

49.

Yoshihara

, Arai

, Hosokawa

, Hagiwara

, Takubo

, Nakamura

, Gomei

, Iwasaki

, Matsuoka

, et al. (2007). Thrombopoietin/MPL Signaling Regulates Hematopoietic Stem Cell Quiescence and Interaction with the Osteoblastic Niche. Cell Stem Cell, 1:685–697.

50.

Nakamura-Ishizu

, Matsumura

, Banu

, Umemoto

and Suda

. (2017). Thrombopoietin-mediated exit from quiescence involves metabolic priming of hematopoietic stem cells for rapid megakaryocyte lineage differentiation. Exp Hematol, 53:S97–SS98.

51.

Gonzalez-Villalva

, Piñon-Zarate

, Falcon-Rodriguez

, Lopez-Valdez

, Bizarro-Nevares

, Rojas-Lemus

, Rendon-Huerta

, Colin-Barenque

and Fortoul

. (2016). Activation of Janus kinase/signal transducers and activators of transcription pathway involved in megakaryocyte proliferation induced by vanadium resembles some aspects of essential thrombocythemia. Toxicol Ind Health, 32:908–918.

52.

Rojnuckarin

, Drachman

and Kaushansky

. (1999). Thrombopoietin-induced activation of the mitogen-activated protein kinase (MAPK) pathway in normal megakaryocytes: role in endomitosis. Blood, 94:1273–1282.

53.

Pulikkan

, Madera

, Xue

, Bradley

, Landrette

, Kuo

, Abbas

, Zhu

, Valk

and Castilla

. (2012). Thrombopoietin/MPL participates in initiating and maintaining RUNX1-ETO acute myeloid leukemia via PI3K/AKT signaling. Blood, 120:868–879.

54.

Yoshida

, Kirito

, Yongzhen

, Ozawa

, Kaushansky

and Komatsu

. (2008). Thrombopoietin (TPO) regulates HIF-1alpha levels through generation of mitochondrial reactive oxygen species. Int J Hematol, 88:43–51.

55.

Ohmizono

, Sakabe

, Kimura

, Tanimukai

, Matsumura

, Miyazaki

, Lyman

and Sonoda

. (1997). Thrombopoietin augments ex vivo expansion of human cord blood-derived hematopoietic progenitors in combination with stem cell factor and flt3 ligand. Leukemia, 11:524–530.

56.

Nishino

, Miyaji

, Ishiwata

, Arai

, Yui

, Asai

, Nakauchi

and Iwama

. (2009). Ex vivo expansion of human hematopoietic stem cells by a small-molecule agonist of c-MPL. Exp Hematol, 37:1364–1377.e4.

57.

Khurana

. (2016). The effects of proliferation and DNA damage on hematopoietic stem cell function determine aging. Dev Dyn, 245:739–750.

58.

Beerman

, Bock

, Garrison

, Smith

, Gu

, Meissner

and Rossi

. (2013). Proliferation-dependent alterations of the DNA methylation landscape underlie hematopoietic stem cell aging. Cell Stem Cell, 12:413–425.

59.

Boward

, Wu

and Dalton

. (2016). Control of cell fate through cell cycle and pluripotency networks. Stem Cells, 34:1427–1436.

60.

Pauklin

and Vallier

. (2013). The cell-cycle state of stem cells determines cell fate propensity. Cell, 155:135–147.

61.

Yuan

, Shen

, Franklin

, Scadden

and Cheng

. (2004). In vivo self-renewing divisions of haematopoietic stem cells are increased in the absence of the early G1-phase inhibitor, p18INK4C. Nat Cell Biol, 6:436–442.

62.

, Yuan

, Shen

and Cheng

. (2006). Hematopoietic stem cell exhaustion impacted by p18 INK4C and p21 Cip1/Waf1 in opposite manners. Blood, 107:1200–1206.

63.

Tian

, Huang

, Gong

, Tian

and Chen

. (2005). Karyotyping, immunophenotyping, and apoptosis analyses on human hematopoietic precursor cells derived from umbilical cord blood following long-term ex vivo expansion. Cancer Genet Cytogenet, 157:33–36.

64.

Alenzi

, Alenazi

, Ahmad

, Salem

, Al-Jabri

and Wyse

. (2009). The haemopoietic stem cell: between apoptosis and self renewal. Yale J Biol Med, 82:7–18.

65.

Janzen

, Fleming

, Waring

, Milne

and Scadden

. (2006). Multifunctional Role of Caspase-3 in Regulating Hematopoietic Stem Cells. Blood, 108:861–861.

66.

Kile

. (2013). The intrinsic apoptosis caspase cascade regulates hematopoietic stem cell homeostasis and function. Exp Hematol, 41:S4.

67.

Sangeetha

, Kale

and Limaye

. (2010). Expansion of cord blood CD34 cells in presence of zVADfmk and zLLYfmk improved their in vitro functionality and in vivo engraftment in NOD/SCID mouse. PLoS One, 5:e12221.

68.

Sangeetha

, Kadekar

, Kale

and Limaye

. (2012). Pharmacological inhibition of caspase and calpain proteases: a novel strategy to enhance the homing responses of cord blood HSPCs during expansion. PLoS One, 7:e29383.

69.

Lin

, Liu

, Shi

, Song

, Huang

, Zou

, Chen

, Li

and Gao

. (2018). Fatty acid oxidation promotes reprogramming by enhancing oxidative phosphorylation and inhibiting protein kinase C. Stem Cell Res Ther, 9:47.

70.

, Gaeta

, Sahakyan

, Chan

, Hong

, Kim

, Braas

, Plath

, Lowry

and Christofk

. (2016). Glycolytic metabolism plays a functional role in regulating human pluripotent stem cell state. Cell Stem Cell, 19:476–490.

71.

Mohrin

, Widjaja

, Liu

, Luo

and Chen

. (2018). The mitochondrial unfolded protein response is activated upon hematopoietic stem cell exit from quiescence. Aging Cell, 17:e12756.

72.

Spencer

, Ferraro

, Roussakis

, Klein

, Wu

, Runnels

, Zaher

, Mortensen

, Alt

, et al. (2014). Direct measurement of local oxygen concentration in the bone marrow of live animals. Nature, 508:269–273.

73.

Takubo

, Nagamatsu

, Kobayashi

, Nakamura-Ishizu

, Kobayashi

, Ikeda

, Goda

, Rahimi

, Johnson

, et al. (2013). Regulation of glycolysis by Pdk functions as a metabolic checkpoint for cell cycle quiescence in hematopoietic stem cells. Cell Stem Cell, 12:49–61.

74.

Srikanth

, Sunitha

, Venkatesh

, Kumar

, Chandrasekhar

, Vengamma

and Sarma

. (2015). Anaerobic glycolysis and HIF1alpha expression in haematopoietic stem cells explains its quiescence nature. J Stem Cells, 10:97–106.

75.

Simsek

, Kocabas

, Zheng

, Deberardinis

, Mahmoud

, Olson

, Schneider

, Zhang

and Sadek

. (2010). The distinct metabolic profile of hematopoietic stem cells reflects their location in a hypoxic niche. Cell Stem Cell, 7:380–390.

76.

Prus

and Fibach

. (2007). The effect of the copper chelator tetraethylenepentamine on reactive oxygen species generation by human hematopoietic progenitor cells. Stem Cells Dev, 16:1053–1056.

77.

Spinazzi

, Sghirlanzoni

, Salviati

and Angelini

. (2014). Impaired copper and iron metabolism in blood cells and muscles of patients affected by copper deficiency myeloneuropathy. Neuropathol Appl Neurobiol, 40:888–898.

78.

Snyder

, Landau

, Rosenheimer

, Mandel

, Glukhman

, Hasson

, Lador

, Olesinski

, Hagler-Price

, et al. (2011). The Stemex Phase II/III Study: challenges in production and delivery of centrally manufactured ex vivo expanded umbilical cord blood (UCB) CD133+ cells to patients with advanced hematological malignancies. Biol Blood Marrow Transplant, 17:S305.

79.

Cimmino

. (2017). Methylation maintains HSC division fate. Proc Natl Acad Sci U S A, 114:192–194.

80.

Trowbridge

, Snow

, Kim

and Orkin

. (2009). DNA methyltransferase 1 is essential for and uniquely regulates hematopoietic stem and progenitor cells. Cell Stem Cell, 5:442–449.

81.

Zhao

, Chen

, Song

, Zhang

, Liu

and Liu

. (2017). Uhrf1 controls the self-renewal versus differentiation of hematopoietic stem cells by epigenetically regulating the cell-division modes. Proc Natl Acad Sci U S A, 114:E142–E151.

82.

Zhang

and Kraus

. (2010). SIRT1-dependent regulation of chromatin and transcription: linking NAD(+) metabolism and signaling to the control of cellular functions. Biochim Biophys Acta, 1804:1666–1675.

83.

Buhrmann

, Busch

, Shayan

and Shakibaei

. (2014). Sirtuin-1 (SIRT1) is required for promoting chondrogenic differentiation of mesenchymal stem cells. J Biol Chem, 289:22048–22062.

84.

Jang

, Huh

, Cho

, Lee

, Park

, Hwang

and Kim

. (2017). SIRT1 Enhances the Survival of Human Embryonic Stem Cells by Promoting DNA Repair. Stem Cell Rep, 9:629–641.

85.

Zhou

, Chen

, Liu

, Zhu

, Si

, Jargstorf

, Li

, Pan

, Gong

, et al. (2018). SIRT1-dependent anti-senescence effects of cell-deposited matrix on human umbilical cord mesenchymal stem cells. J Tissue Eng Regen Med, 12:e1008–e1021:e1008.

86.

Yang

, Bi

, Xue

, Wang

, Lu

, Zhang

, Chen

, Chu

, Yang

, Wang

and Liu

. (2015). Multifaceted Modulation of SIRT1 in Cancer and Inflammation. Crit Rev Oncog, 20:49–64.

87.

Iwata

, Ogata

, Okumura

and Taguchi

. (2003). Induction of differentiation in human promyelocytic leukemia HL-60 cell line by niacin-related compounds. Biosci Biotechnol Biochem, 67:1132–1135.

88.

Peled

, Shoham

, Aschengrau

, Yackoubov

, Frei

, Rosenheimer

, Lerrer

, Cohen

, Nagler

, Fibach

and Peled

. (2012). Nicotinamide, a SIRT1 inhibitor, inhibits differentiation and facilitates expansion of hematopoietic progenitor cells with enhanced bone marrow homing and engraftment. Exp Hematol, 40:342–55.e1.

89.

Wright

. (2014). Exercise- and resveratrol-mediated alterations in adipose tissue metabolism. Appl Physiol Nutr Metab, 39:109–116.

90.

Palomer

, Capdevila-Busquets

, Alvarez-Guardia

, Barroso

, Pallàs

, Camins

, Davidson

, Planavila

, Villarroya

and Vázquez-Carrera

. (2013). Resveratrol induces nuclear factor-kappaB activity in human cardiac cells. Int J Cardiol, 167:2507–2516.

91.

Wang

, Wang

, Jia

, Liu

, Shu

and Liu

. (2016). Resveratrol Increases Anti-Proliferative Activity of Bestatin Through Downregulating P-Glycoprotein Expression Via Inhibiting PI3K/Akt/mTOR Pathway in K562/ADR Cells. J Cell Biochem, 117:1233–1239.

92.

Kutil

, Temml

, Maghradze

and Pribylova

. (2014). Impact of wines and wine constituents on cyclooxygenase-1, cyclooxygenase-2, and 5-lipoxygenase catalytic activity. Mediators Inflamm, 2014:178931.

93.

Heinz

, Ehrnström

, Schambach

, Schwarzer

, Modlich

and Schiedlmeier

. (2015). Comparison of different cytokine conditions reveals resveratrol as a new molecule for ex vivo cultivation of cord blood-derived hematopoietic stem cells. Stem Cells Transl Med, 4:1064–1072.

94.

Berman

, Motechin

, Wiesenfeld

and Holz

. (2017). The therapeutic potential of resveratrol: a review of clinical trials. NPJ Precis Oncol, 1::35.

95.

Perez-Campo

, Borrow

, Kouskoff

and Lacaud

. (2009). The histone acetyl transferase activity of monocytic leukemia zinc finger is critical for the proliferation of hematopoietic precursors. Blood, 113:4866–4874.

96.

Attema

, Papathanasiou

, Forsberg

, Xu

, Smale

and Weissman

. (2007). Epigenetic characterization of hematopoietic stem cell differentiation using miniChIP and bisulfite sequencing analysis. Proc Natl Acad Sci U S A, 104:12371–12376.

97.

Nishino

, Wang

, Mochizuki-Kashio

, Osawa

, Nakauchi

and Iwama

. (2011). Ex vivo expansion of human hematopoietic stem cells by garcinol, a potent inhibitor of histone acetyltransferase. PLoS One, 6:e24298.

98.

Araki

, Yoshinaga

, Boccuni

, Zhao

, Hoffman

and Mahmud

. (2007). Chromatin-modifying agents permit human hematopoietic stem cells to undergo multiple cell divisions while retaining their repopulating potential. Blood, 109:3570–3578.

99.

Mahmud

, Petro

, Baluchamy

, Li

, Taioli

, Lavelle

, Quigley

, Suphangul

and Araki

. (2014). Differential effects of epigenetic modifiers on the expansion and maintenance of human cord blood stem/progenitor cells. Biol Blood Marrow Transplant, 20:480–489.

100.

Chaurasia

, Gajzer

, Schaniel

, D'Souza

and Hoffman

. (2014). Epigenetic reprogramming induces the expansion of cord blood stem cells. J Clin Invest, 124:2378–2395.

101.

Fares

, Chagraoui

, Gareau

, Gingras

, Ruel

, Mayotte

, Csaszar

, Knapp

, Miller

, et al. (2014). Pyrimidoindole derivatives are agonists of human hematopoietic stem cell self-renewal. Science, 345:1509–1512.

102.

Fares

, Chagraoui

, Lehnertz

, MacRae

, Mayotte

, Tomellini

, Aubert

, Roux

and Sauvageau

. (2017). EPCR expression marks UM171-expanded CD34(+) cord blood stem cells. Blood, 129:3344–3351.

103.

Martin

and Park

. (2017). EPCR: a novel marker of cultured cord blood HSCs. Blood, 129:3279–3280.

104.

, Xia

, Wang

, Liu

, Zhao

, Yang

, Hu

, Zhang

, Huang

, et al. (2017). Pyrimidoindole derivative UM171 enhances derivation of hematopoietic progenitor cells from human pluripotent stem cells. Stem Cell Res, 21:32–39.

105.

Cohen

, Roy

, Lachance

, Marinier

, Delisle

, Roy

, Busque

, Ahmad

, Bambace

, et al. (2017). Single UM171 expanded cord blood transplant is feasible and safe, accelerates engraftment, reduces hospitalization length and most importantly improves HLA matching. Exp Hematol, 53:S48–S49.

106.

Schmid

, Snoek

, Fröhli

, van der Bent

, Kammenga

and Hajnal

. (2015). Systemic Regulation of RAS/MAPK Signaling by the Serotonin Metabolite 5-HIAA. PLoS Genet, 11:e1005236.

107.

Hadden

, Fahmi

, Cooper

, Savenka

, Lupashin

, Roberts

, Maroteaux

, Hauguel-de Mouzon

and Kilic

. (2017). Serotonin transporter protects the placental cells against apoptosis in caspase 3-independent pathway. J Cell Physiol, 232:3520–3529.

108.

Gurbuz

, Asoglu

, Ashour

, Salama

, Kilic

and Ozpolat

. (2016). A selective serotonin 5-HT1B receptor inhibition suppresses cells proliferation and induces apoptosis in human uterine leiomyoma cells. Eur J Obstet Gynecol Reprod Biol, 206:114–119.

109.

Ori

, De Lucchini

, Marras

and Nardi

. (2013). Unraveling new roles for serotonin receptor 2B in development: key findings from Xenopus. Int J Dev Biol, 57:707–714.

110.

Stoyek

, Jonz

, Smith

and Croll

. (2017). Distribution and chronotropic effects of serotonin in the zebrafish heart. Auton Neurosci, 206:43–50.

111.

Yang

, Srikiatkhachorn

, Anthony

and Chong

. (1996). Serotonin stimulates megakaryocytopoiesis via the 5-HT2 receptor. Blood Coagul Fibrinolysis, 7:127–133.

112.

Yang

, Li

, Ng

, Chuen

, Lau

, Cheng

, Liu

, Li

, Yuen

, et al. (2007). Promoting effects of serotonin on hematopoiesis: ex vivo expansion of cord blood CD34+ stem/progenitor cells, proliferation of bone marrow stromal cells, and antiapoptosis. Stem Cells, 25:1800–1806.

113.

Nebigil

, Etienne

, Messaddeq

and Maroteaux

. (2003). Serotonin is a novel survival factor of cardiomyocytes: mitochondria as a target of 5-HT2B receptor signaling. FASEB J, 17:1373–1375.

114.

Hsiung

, Tamir

, Franke

and Liu

. (2005). Roles of extracellular signal-regulated kinase and Akt signaling in coordinating nuclear transcription factor-kappaB-dependent cell survival after serotonin 1A receptor activation. J Neurochem, 95:1653–1666.