Getting more for your marrow: boosting hematopoietic stem cell numbers with PGE₂

Abstract

Throughout the lifetime of an individual, hematopoietic stem cells (HSCs) self-renew and differentiate into lineages that include erythrocytes, platelets and all immune cells. HSC transplantation offers a potentially curative treatment for a number of hematopoietic and non-hematopoietic malignancies as well as immune and genetic disorders. Limited availability of immune-matched donors reduces the viable options for many patients in need of HSC transplantation, particularly those of diverse racial and ethnic backgrounds. Due to rapid availability and less stringent immune-matching requirements, umbilical cord blood (UCB) has emerged as a valuable source of transplantable HSCs. A single UCB unit contains a suboptimal number of HSCs for treating larger children or adults and there has thus been great clinical interest in expanding UCB HSCs ex vivo for use in transplantation. In this review we discuss the latest research and future avenues for the therapeutic use of small lipid mediator dmPGE₂ to expand HSC numbers for transplantation. Originally identified in a chemical screen in zebrafish, dmPGE₂ has now advanced to a phase II clinical trial as a therapy for patients with leukemia and lymphoma who are undergoing UCB transplantation.

Introduction

Hematopoietic stem cells (HSCs) are a rare population of self-renewing progenitors that maintain life-long production of all blood and immune cell lineages¹. HSCs are of outstanding interest to clinical researchers for many reasons. The transplantation of these cells can be used to treat a variety of human diseases, including numerous cancers and a wide range of blood and immune disorders². Transplantable HSCs can be obtained from three sources: bone marrow, peripheral blood and umbilical cord blood (UCB)³. Historically, bone marrow has been the standard source of HSCs for transplantation. Recently, it is becoming more common that HSCs are pharmacologically mobilized out of the marrow and then isolated from peripheral blood – an extraction procedure that is far less invasive.

The use of one's own stem cells, known as autologous transplantation, is the preferred method as it avoids immune differences that can lead to delayed engraftment, graft-versus-host disease and graft rejection³. This is often not possible, however, as it requires that healthy HSCs be obtained at an earlier date (e.g. pre-malignancy). The use of donor HSCs, known as allogenic transplantation, can work well if an HLA-matched donor is available. UCB serves as an attractive alternative source of HSCs for transplantation because the naïve state of immune cells within UCB reduces the stringency of immune-matching requirements⁴. UCB can be cryopreserved and stored in cord blood banks and is therefore more readily available. Although a rich source of HSCs, a single UCB unit contains an insufficient number of cells for treating larger children and adults. It is generally accepted that the number of transplanted HSCs directly correlates with the likelihood of successful engraftment and reduced time to immune recovery. It is therefore standard practice that two UCB units are administered, which puts a tremendous strain on cord blood bank resources. The use of two UCB units is also complicated by competition between the two cord bloods – one unit typically dominates by three months after the transplant⁴. The ability to expand the number of HSCs in a single UCB unit prior to transplantation would thus have great clinical significance.

Strategies to expand HSCs for transplantation

In recent years there have been a number of different strategies to expand UCB HSCs ex vivo for use in transplantation^5,6. These efforts have in large part, however, seen limited success in terms of attaining clinically relevant numbers of cells. Expanding HSCs in culture requires maintaining the self-renewal properties of the cells while preventing their differentiation. These seemingly simple objectives have proven challenging to accomplish in a dish, when HSCs are outside of the in vivo microenvironment. Initial strategies to expand UCB HSCs ex vivo involved the addition of hematopoietic cytokines to the culture media, including factors such as SCF, Tpo, Flt-3L, IL-3, IL-6 and G-CSF⁶. Although the extent to which these factors enhance expansion is limited, it is worth noting that these so-called cytokine cocktails still serve as the basal media for many current HSC expansion protocols.

Recently, a number of small molecules have been shown to support HSC expansion. These include the ayrl hydrocarbon receptor agonist SR1⁷, the copper chelator TEPA⁸, the GSK-3β inhibitor BIO⁹, NR-101¹⁰ and prostaglandin E₂ (PGE₂)¹¹. Interestingly, some of these compounds function as surrogates for the hematopoietic cytokines mentioned above. NR-101, for instance, binds and activates the Tpo receptor c-MPL and is even more effective at expanding HSCs than Tpo¹⁰. Compared to larger protein-based molecules, these compounds offer several advantages as a method to modulate HSC numbers¹². Small molecules are easier and cheaper to manufacture, and the window of treatment with small molecules can be tightly controlled. Chemicals can be removed, for example, before treated cells are introduced into patients – avoiding permanent modifications to the HSCs (e.g. retroviral gene insertion) that could potentially lead to dis-regulated proliferation and/or malignancy.

Other strategies the expand HSCs have been modeled more directly after the in vivo niche or the mechanisms that regulate HSC formation during development. Co-culturing HSCs with supportive cell types that are present in the bone marrow niche, such as mesenchymal stem cells, can enhance HSC expansion and improve the engraftment of UCB stem cells upon transplantation¹³. Modulation of certain developmental signaling pathways have also been shown to promote robust HSC expansion. Addition of soluble Wnt protein to the media increases HSC numbers and improves engraftment when the stem cells are transplanted into recipient animals¹⁴. Similarly, culturing UCB stem cells in the presence of an immobilized Notch ligand enhances HSC expansion and improves transplantability¹⁵. These latter approaches, targeting pathways that regulate HSC formation during normal development, have, to date, resulted in some of the best yields in ex vivo HSC expansion.

In this review we discuss the recent use of dmPGE₂ (a stable derivative of prostaglandin E₂) as a new strategy for HSC expansion that targets a developmental regulatory pathway while affording the therapeutic benefits of a small molecule. After just a brief ex vivo pulse of dmPGE₂, long-lasting effects on HSC function have been observed without over-proliferation side effects. dmPGE₂ was originally identified in a chemical screen in zebrafish and has now has now advanced to phase II clinical trials for UCB transplantation in patients with leukemia and lymphoma.

PGE₂ synthesis, reception and downstream intracellular signaling

Prostaglandins are members of the eiconsanoid family of lipid compounds – molecules that play major roles in inflammation and immune response, as well as regulate a number of physiological tissue responses such as blood pressure and stomach acid production¹⁶. Prostaglandins are synthesized by the cyclooxygenases Cox1 and Cox2, through oxidation of the derivative arachidonic acid to form PGG₂¹⁷. PGG₂ is then reduced to PGH₂, the molecule from which all three classes of prostaglandins are made. In general, Cox1 is more ubiquitously expressed, whereas Cox2 is silenced under normal physiology, but can be rapidly induced, for example, during times of stress.

Prostaglandins are not stored, but rather synthesized as needed and function locally as paracrine or autocrine lipid mediators, having a half-life of about one minute in circulation¹⁶. A number of different cell types produce PGE₂ and it can be found within most tissues throughout the body. PGE₂ can signal through four G-protein coupled E-prostanoid receptors PTGER1-4 (EP1-4), which, in contrast to PGE₂ itself, are expressed in more tissue-restricted patterns¹⁸. Activation of EP1, EP2 and EP4 result in positive signal transduction through either mobilization of intracellular Ca2+ (EP1) or stimulation of cAMP production (EP2 and EP4). Binding to EP3, on the other hand, acts to negatively regulate cAMP.

A chemical screen in zebrafish identifies PGE₂ as a conserved regulator of vertebrate HSC homeostasis

In 2007 North et al. conducted a high-throughput chemical screen for small molecules that could modulate vertebrate HSC homeostasis¹¹. Out of a library of nearly 2,500 biologically active compounds, the authors identified 82 chemicals that either increased or decreased the expression of runx1 and cmyb (conserved HSC markers) in zebrafish embryos. Surprisingly, 10 of the identified compounds affected prostaglandin signaling. When embryos were treated with a long-acting derivative of PGE₂, 16,16-dimethyl-PGE₂ (dmPGE₂), a marked increase in HSCs numbers was observed in the aorta-gonad-mesonephros region (AGM) – the tissue from which definitive HSCs arise during development (Fig. 1A). Injection of morpholinos targeting Cox1 and Cox2 (the enzymes responsible for PGE₂ synthesis) or treatment with the Cox inhibitor indomethacin led to reduced runx1/cmyb staining in the AGM (Fig. 1A), indicating that endogenous prostaglandin signaling is necessary for HSC formation. Exposure to dmPGE₂ was also shown to enhance the marrow recovery of adult fish following a sub-lethal dose of irradiation. The authors went on to demonstrate that treatment with dmPGE₂ could amplify multi-potent progenitors in murine embryonic stem cell differentiation assays and enhance murine spleen colony forming units 12-days post-transplant. Limit dilution analysis in mice showed that treatment with dmPGE₂ also increased the frequency of long-term repopulating HSCs. Collectively, these studies demonstrated that PGE₂ was a conserved regulator of vertebrate HSC homeostasis.

Figure 1. dmPGE₂ regulates vertebrate HSC function.

(A) A schematic representation depicts the basic workflow for the zebrafish chemical screen that identified dmPGE₂ and an endogenous role for prostaglandin signaling in HSC formation. Adult zebrafish were in-crossed and collected embryos were treated with chemicals from the 3-somite stage to 36 hours post fertilization. Whole embryo in situ hybridization to runx1/cmyb (conserved HSC markers) was then used to examine HSC formation. Embryos treated with the stable derivative dmPGE₂ displayed increased runx1/cmyb staining in the AGM whereas treatment with the Cox inhibitor indomethacin led to reduced HSC formation. (B) 2-hour ex vivo pulse treatment with dmPGE₂ leads to increases in HSC function that last for as long as five serial transplantations. (C) Prolonged in vivo exposure to dmPGE₂ leads to enhanced engraftment; however, the treated cells eventually exhaust and lose differentiation potential. (D) dmPGE₂ signals through EP2 and EP4 receptors and an interaction with Wnt signaling. Downstream transcriptional changes lead to improved homing, survival and proliferation of the HSCs.

Although unexpected, this work was not the first time PGE₂ had been linked to hematopoiesis. As far back as the 1970s, it had been documented that unmodified PGE₂ had effects on colony forming unit-spleen (CFU-S) stem cells and on more lineage-restricted hematopoietic progenitors using in vitro colony-forming assays^19–21. These earlier studies, however, produced varying and sometimes conflicting results and in the two decades that followed, few new insights had come about. These new findings in the fish not only revitalized interest in the area but also provided strong evidence that dmPGE₂ could provide an effective means to boost stem cell numbers in a transplantation setting.

dmPGE₂ signals through EP2/EP4 and an interaction with the Wnt pathway

In zebrafish, Ptger2 (EP2) and Ptger4 (EP4) are the PGE₂ receptors expressed on HSCs. Knockdown of ptger2 or ptger4 results in a decrease of runx/cmyb expression in the AGM that is not rescued with PGE₂ treatment¹¹. This suggests that PGE₂ signals through Ptger2 and Ptger4 to regulate HSC formation in zebrafish embryos. Interestingly, it had been demonstrated in previous biochemical studies that binding of PGE₂ to EP2 or EP4 can lead to activation of the GSK-3/β-catenin signaling pathway via protein kinase A and phosphoinositide 3-kinase, respectively²². Additional work in the zebrafish by Goessling et al., demonstrated an in vivo PGE₂-Wnt interaction. PGE₂ enhanced the effect of Wnt signaling during embryogenesis by stabilizing β-catenin, and was required for Wnt-mediated regulation of HSC development (Fig. 1D)²³.

All four EP receptors are expressed on murine progenitor populations (LSK) as well as more purified stem cell populations (LSKCD48⁻CD150⁺). EP1–EP4 receptors are also found on human CD34⁺CD38⁻ cells²⁴. This data, combined with the observed genetic interaction between PGE₂ and Wnt, suggests the EP2 and EP4 receptors in particular play an important role in the dmPGE₂-mediated regulation of HSCs. Knockdown of EP4 in LSK cells results in decreased repopulation ability following transplantation, as well as a skewing towards T cell and myeloid lineages during differentiation²⁵. In contrast, EP2 knockout mice exhibit normal hematopoiesis, reconstitution capabilities, and differentiation. In addition, treatment of LSK cells with an EP4 agonist, but not an EP2 agonist, lead to an increase in phosphorylation of GSK3β and β-catenin. Co-treatment of a PKA inhibitor with PGE₂ blocks the activation of GSK3β. Together, this data suggests that PGE₂ acts through EP4 to activate the cAMP/PKA pathway in HSCs.

dmPGE₂ improves engraftment by enhancing HSC homing, survival and proliferation

The aforementioned studies implicated particular EP receptors and an interaction with the Wnt pathway, but the transcriptional changes occurring downstream of dmPGE₂ treatment, through which HSC dynamics were being affected, remained incompletely understood. More recent studies have provided additional mechanistic insight into the stem-cell-enhancing activity of dmPGE₂. In 2009, a study by Hoggatt et al. reported that ex vivo treatment of murine whole bone marrow (WBM) with dmPGE₂ improved engraftment, likely through an enhancement in HSC homing (via up-regulation of CXCR4) and survival (via up-regulation of Survivin and down-regulation of caspase-3) (Fig. 1B and D)²⁴. The improvement in HSC function was maintained in secondary transplantation, without additional dmPGE₂ treatment, suggesting there was an effect on long-term (LT)-HSCs.

These initial findings have since been confirmed by additional studies looking at global gene expression patterns in response to dmPGE₂ treatment^26,27. PGE₂ seems to both activate general cell survival and proliferation pathways but also lead to a specific up-regulation in cytokine response. Interestingly, recent work has implicated HSCs as potent secretors of cytokines in response to infectious stimuli²⁸. Treating HSCs with dmPGE₂ could therefore activate the endogenous hematopoietic regeneration program that is turned on during injury. Consistent with the dmPGE₂-induced improvement in HSC homing, it was recently shown that blocking prostaglandin signaling can lead to HSC mobilization²⁹. The exact temporal requirement of different cell migration and survival pathways, as well as the interplay with the niche during UCB transplantation, remains to be determined for dmPGE₂ treatment.

The cellular context, culture conditions and dose of dmPGE₂ are important

A common theme to emerge from recent studies is that variations in the dose of dmPGE₂ or the duration of exposure can have significantly different effects on HSC dynamics. This is likely, in part, why early studies with PGE₂ often gave varying and sometimes conflicting results. A short 2-hour ex vivo pulse on ice was shown to be sufficient to increase HSC numbers and enhance engraftment of treated cells upon transplantation into mice^11,24. As part of a recent phase I clinical study, Cutler et al. used expression profiling to conduct an extensive analysis of various dmPGE₂ treatment conditions²⁶. It was concluded that a 2-hour pulse of 10 μM dmPGE₂ at 37°C in nutrient-free media were the optimal conditions for treating UCB cells ex vivo in a clinical transplantation setting.

Work by Frisch et al. provided the first data on the effects of in vivo dmPGE₂ treatment in mice³⁰. The authors adopted a PGE₂ treatment protocol previously used in rats. dmPGE₂ was delivered by intraperitoneal injection (6 mg/kg), twice daily for 16 days (Fig. 1C). Similar to the in vitro data, a significant increase in LSK cell numbers was observed without adversely affecting hematopoietic differentiation. SLAM receptors were used to differentiate long-term HSCs (LT-HSCs) from multi-potent progenitors (MPPs) and short-term HSCs (ST-HSCs) and it was found that in vivo dmPGE₂ treatment expanded the ST-HSC/MPP subpopulation without changing the frequency of LT-HSCs. Cells transplanted from these in vivo-treated mice displayed increased engraftment and hematopoietic reconstitution in recipient animals; however, the competitive advantage was eventually lost at six weeks post-transplantation and in secondary transplants no significant difference was observed. These experiments suggested that long-term in vivo exposure to dmPGE₂ was having a different effect on HSCs compared to the short ex vivo pulse treatments.

Continuous exposure to dmPGE₂ may induce multiple rounds of excessive proliferation from which cells cannot recover; it may also lead to receptor down-regulation, effectively changing the responsiveness of the cells to dmPGE₂. Supporting this notion, data from cell culture experiments has shown that long-term treatment with dmPGE₂ results in down-regulation of EP2 and EP4 receptors³¹ and patients with mutations in the prostaglandin transporter gene, SLCO2A1, have elevated levels of circulating PGE₂ and experience pancytopenia with bone marrow hypocellularity³².

In zebrafish, mouse and humans, components of the prostaglandin pathway and PGE₂ receptors are present on other cells types in addition to HSCs, such as endothelial cells, stromal cells and leukocytes^16,33. It is likely that in vivo dmPGE₂ treatments also affect these other cells that then indirectly influence HSCs. Consistent with this, studies from the Calvi Lab have documented changes in trabecular bone microarchitecture and macrophage numbers following in vivo treatment with dmPGE₂^30,34. In addition, the Lapidot Lab demonstrated that in vivo and in vitro treatment with dmPGE₂ increased expression of CXCL12 on a population of nestin⁺ stromal cells that are adjacent to HSPCs and play a role in their preservation³⁵. As many of the HSC sources used in ex vivo studies are heterogeneous cell populations (e.g. WBM, CD34+ and LSK cells), it is possible that similar non-cell autonomous effects could occur in these contexts. Interestingly, it was recently published that dmPGE₂ promotes the survival of naïve UCB T cells through the Wnt/β-catenin pathway³⁶. This protective effect could enhance recipient T-cell chimerism following transplant, which would have significant clinical implications given that delayed immunological reconstitution is a huge risk factor for transplant patients.

Completion of a Phase I clinical trial for UCB transplantation

In 2010 preclinical studies were performed to evaluate the therapeutic potential of dmPGE₂ in UCB transplantation²⁷. In this work, human and non-human primate HSCs were treated with dmPGE₂. Treatment with dmPGE₂ improved engraftment of human HSCs upon xenotransplantation into NOD/SCID mice. dmPGE₂ treatment was also evaluated in autologous transplantation of HSCs from mobilized peripheral blood in non-human primates and stable engraftment was observed up to one year later. Together these results indicated that PGE₂ has evolutionarily conserved functions in regulating HSC numbers and provided sufficient preclinical data to move forward with an FDA-approved phase I clinical trial.

The fact that PGE₂ was already being used for various purposes in the clinic facilitated the transition to phase I clinical studies. A two-year trial was undertaken at Dana Farber Cancer Center and Massachusetts General Hospital to examine the safety of ex vivo dmPGE₂ treatment to expand UCB HSC for transplantation in human patients²⁶. The trial enrolled 12 patients that were undergoing treatment for leukemia or lymphoma. Each patient received two UCB units that were infused in succession – the first being treated with dmPGE₂ and the second being untreated (Fig. 2). Neutrophil engraftment was observed in 100% of patients in the trial, compared to 90% of historic control and the median time to engraftment was 17.5 days versus 21 days, respectively. dmPGE₂-treated UCB displayed enhanced engraftment by outcompeting the untreated UCB unit in 10 out of 12 patients. These initial results demonstrate that this dmPGE₂ treatment regimen is clinically well tolerated and on the basis of these results patients are now being enrolled in a randomized phase II clinical trial to evaluate the efficacy of dmPGE₂ treatment. These studies will involve 40–80 patients and will be conducted at multiple centers. In addition, new phase I studies have been initiated to examine the use of dmPGE₂ in single UCB transplantation and in autologous peripheral blood HSC transplantation.

Figure 2. The clinical application of dmPGE₂ to improve UCB transplantation.

The schematic on the left depicts the standard protocol in which two UCB units are administered to the patient. The clinical strategy for using dmPGE₂ to improve UCB transplantation is shown on the right. The first UCB unit is treated with a 2-hour ex vivo pulse of dmPGE₂ before infusion into the patient. A second untreated UCB is administered in succession. Clinical outcomes for each group are listed at the bottom of the figure.

Directions for future studies with dmPGE₂

Significant advances have been made towards understanding how dmPGE₂ affects HSCs and initial studies suggest it may be possible in the not-too-distant future to sufficiently expand the number of HSCs in a single UCB unit for transplantation. At the same time there remain many important questions for future research. Fully elucidating the downstream transcriptional changes that occur following dmPGE₂ treatment will be crucial for better understanding how this compound exerts its effects on HSCs. Genomic approaches like the one used by Cutler et al. could be performed on individual HSCs or on a subpopulation basis (e.g. on cycling versus quiescent cells or on sorted MPPs, ST-HSCs and LT-HSCs).

It was recently shown that short 2-hour ex vivo pulse treatment of murine HSCs with dmPGE₂ can have effects that remarkably last for as long as five serial transplantations without malignant transformation (Fig. 1B)³⁷. On a cell-by-cell basis, however, the HSCs were not inherently more competitive, suggesting the observed effects may be due to an increase in HSC numbers through proliferation or a conversion in HSC fate (e.g. a change in ST-HSC fate to a more LT-HSC phenotype). Such changes could be due, in part, to alterations in the epigenetic state of the cells. PGE₂ has been shown to affect DNA methylation patterns in the intestine and increase global DNA methylation via induction of DNMT3a in fibroblasts³⁸. It will be interesting in future studies to explore potential roles for epigenetic modifiers and to examine DNA methylation patterns before, during and after treatment with dmPGE₂. As a clearer picture of the downstream cellular mechanisms emerges, so will new therapeutic opportunities to enhance the positive effects of dmPGE₂ on HSCs or to alleviate medical conditions in which PGE₂ levels are aberrant.

Exploring additional UCB stem cell expansion protocols

In a recent clinical trial it was shown that co-culturing UCB cells with mesenchymal stem cells leads to a 40-fold increase in transplantable CD34+ cells¹³. A similar trial demonstrated that culturing UCB cells for 14 days in the presence of the Notch ligand Delta could expand stem cells numbers and that these cells could then be infused with a second UCB unit¹⁵. In both of these cases, however, the expanded stem cells were unable to contribute to long-term hematopoiesis and were eventually lost. Also being evaluated in a clinical trial is the small molecule StemRegenin1 (SR1), which was shown to have remarkable effects on the expansion of CD34+ cells⁷. Similar to dmPGE₂, treatment with SR1 prior to transplantation increased the frequency of short-term repopulating units without affecting long-term engraftment or multi-lineage reconstitution ability. It has become clear that crosstalk between signaling pathways regulates HSC formation and homeostasis. Treatment with the GSK-3β inhibitor BIO (a Wnt signaling activator), for example, leads to activation of Notch in UCB cells and may promote Notch signaling during ex vivo expansion⁹. Given that PGE₂ and Wnt signaling pathways interact to regulate HSC function²³, it seems likely that targeting additional pathways and/or adapting a co-culture-type expansion protocol, in combination with dmPGE₂ treatment, will be more effective. Interestingly, both SR1 and dmPGE₂ were used in a recent study to better preserve HSCs such that the cells could be cultured long enough for targeted genome editing³⁹. Future approaches involving the integration of multiple different strategies are likely provide the most effective means to enhance the ex vivo expansion of UCB stem cells for clinical transplantation.