REVIEW URRENT C OPINION
Role of epigenetic reprogramming in hematopoietic stem cell function Camelia Iancu-Rubin and Ronald Hoffman
Purpose of review Epigenetic regulatory networks determine the fate of dividing hematopoietic stem cells (HSCs). Prior attempts at the ex-vivo expansion of transplantable human HSCs have led to the depletion or at best maintenance of the numbers of HSCs because of the epigenetic events that silence the HSC geneexpression pattern. The purpose of this review is to outline the recent efforts to use small molecules to reprogram cultured CD34þ cells so as to expand their numbers. Recent findings Chromatin-modifying agents (CMAs) reactivate the gene-expression patterns of HSCs that have been silenced as they divide ex vivo. Increasing evidence indicates that CMAs act not only by promoting HSC symmetrical self-renewal divisions, but also by reprogramming progenitor cells, resulting in greater numbers of HSCs. The use of such CMAs for these purposes has not resulted in malignant transformation of the ex-vivo treated cell product. Summary The silencing of the gene-expression program that determines HSC function after ex-vivo culture can be reversed by reprogramming the progeny of dividing HSCs with transient exposure to CMAs. The successful implementation of this approach provides a strategy which might lead to the development of a clinically relevant means of manufacturing increased numbers of HSCs. Keywords epigenetic reprogramming, hematopoietic stem cell expansion, hematopoietic stem cell transplantation
INTRODUCTION
single UCB graft did not lead to a survival advantage in either children or adolescents [2 ]. These observations have led to renewed interest in creating exvivo strategies to expand the number of HSCs within a single UCB collection. Such efforts are not only of clinical importance, but also would lead to a greater understanding of the events that determine the fate of dividing HSCs. &
Hematopoietic stem cell (HSC) transplantation provides patients with refractory hematological malignancies or genetic disorders due to blood cell abnormalities an opportunity for cure. Lack of access to a matched histocompatible allogeneic sibling, unrelated matched HSC donor or haploidentical donor prevents many such patients from receiving such therapy [1]. The identification of unrelated umbilical cord blood (UCB) as a readily available source of such normal HSC grafts has provided another alternative HSC source. As there are a fixed, limited number of HSCs within a single UCB collection, the clinical outcomes of adults receiving such grafts have been less favorable than that observed in children receiving UCB collections [1]. Such limitations have resulted in the infusion of two different matched UCB collections in order to increase the number of HSCs. Although several phase 2 trials have provided promising results, a more extensive phase 3 trial concluded that transplantation with two UCB units compared with a
PRIOR ATTEMPTS TO EXPAND HEMATOPOIETIC STEM CELLS The initial efforts to expand HSCs ex vivo attempted to recreate a hematopoietic marrow microenvironment The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA Correspondence to Ronald Hoffman, MD, Division of Hematology and Medical Oncology, Box 1079, 1 Gustave L. Levy Place, New York, NY 10029, USA. Tel: +1 212 241 2296; e-mail: [email protected] Curr Opin Hematol 2015, 22:279–285 DOI:10.1097/MOH.0000000000000143
1065-6251 Copyright ß 2015 Wolters Kluwer Health, Inc. All rights reserved.
www.co-hematology.com
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Hematopoiesis
KEY POINTS Epigenetic regulation is a key factor determining the HSC fate decision. Chromatin-modifying agents can promote HSC expansion by inducing HSC symmetrical self-renewal and by reprogramming HPC to HSC. HSC grafts expanded by means of epigenetic reprogramming are a potential source for allogeneic HSC transplantation for patients with refractory hematological malignancies or genetic blood disorders.
which favored symmetrical stem cell expansion [3– 9]. For these purposes, combinations of cytokines known to be elaborated by marrow microenvironmental cells and to promote HSC cycling and their subsequent division were added. Such efforts, however, resulted in the generation of large numbers of hematopoietic progenitor cells (HPCs) and precursor cells but reduced numbers of HSCs. The rapid ex-vivo cycling and division of cord blood CD34þ cells that occurred in the presence of such cytokine combinations led to HSC commitment, with the residual marrow-repopulating potential being attributed to a small fraction of HSCs that remained quiescent or had undergone a limited number of cell divisions. Mesenchymal cell feeder layers or a number of molecules such as immobilized notch ligand, a copper chelator, an aryl hydrocarbon receptor antagonist (SR1), prostaglandin E2 (PGE2), and a pyrimidoindole derivative (UM171) have been added to such cytokine combinations with the hope of further expanding the number of transplantable cord blood HSCs [3–8,10]. To date, these attempts have met with limited success toward clinical applications. The reconstruction of a marrow microenvironment that favors stem HSC expansion ex vivo appears to be an elusive goal as the components of the niche which favor HSC self-renewal remain uncertain and the ability of bioengineers to construct such a dynamic and complex environment which would be sustainable ex vivo for prolonged periods of time would require overcoming numerous obstacles.
STEM CELL DECISIONS AND EPIGENETIC MODIFICATIONS HSCs are able to balance the self-renewal with commitment in vivo by controlling the proportion of asymmetric and symmetric cell divisions that they undergo, but this balance is lost following ex-vivo culture (Fig. 1). During asymmetric HSC division, one daughter cell remains an HSC identical to the mother cell, whereas the other becomes committed 280
www.co-hematology.com
and develops into a HPC. By contrast, during symmetrical division a HSC can divide to generate either two HSCs (symmetrical renewal) or two committed HPCs (symmetrical commitment). Asymmetrical HSC division results in the maintenance of the numbers of HSCs while allowing for a progressive increase in the numbers of HPCs. Symmetrical renewal divisions, by contrast, lead to an expansion of the pool of HSCs, whereas symmetrical commitment divisions result in the generation of differentiated cells and eventual HSC exhaustion. A growing number of investigators have speculated that dynamic epigenetic events alter the chromatin structure and lead to transcriptional programs that determine the HSC status [11,12 ,13–17]. In vivo, these epigenetic events likely occur in response to a variety of microenvironmental cues which change during development or occur in response to external stimuli or demands requiring alterations in blood cell production. Gene expression is dynamically regulated by the modifications of chromatin which occurs largely as a consequence of DNA methylation and histone acetylation [12 ]. Several groups, including our own, have used chromatin-modifying agents (CMAs) to treat HSCs in vitro in order to favor the expression of genetic programs which support symmetrical renewal divisions or to reprogram primitive HPCs back to HSCs [18,19,20 ,21–23,24 ,25,26 , 27–33]. Ex-vivo culture conditions represent stressful conditions which frequently lead to HSC commitment. As HSCs are slow cycling cells, a prolonged period of culture would be needed if the expansion of HSCs as entirely dependent on a significant fraction of HSCs to undergo symmetrical self-renewing divisions. The period of ex-vivo treatment could be theoretically shortened if the agent or agents used to expand HSCs were also capable of reprogramming more differentiated HPCs. Cellular reprogramming induces differentiated cells to revert back to undifferentiated cells. The epigenetic barrier for reprogramming mammalian cells has been overcome with the use of reprogramming factors such a Oct3, Oct4, Sox2, Klf4, Myc, and Lin28 [14]. Such an approach was utilized by Takahashi and Yamanaka [34] to create immortalized pluripotent stem cells (iPSCs). This approach, however, is not currently suitable to generate larger numbers of transplantable HSCs because of the safety concerns associated with their propensity to undergo maligant transformation (i.e. formation of teratomas) and the limited engaftment potential of iPSC-derived cells with an HSC phenotype. For the strategy of reprogramming of UCB HSCs to be clinically applicable, it should be not only capable of promoting the division of primitive HSC populations, but also capable of transiently &&
&&
&&
&&
&
Volume 22 Number 4 July 2015
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Role of epigenetic reprogramming in hsc Iancu-Rubin and Hoffman
Symmetrical
Asymmetrical
HSC
Symmetrical
HSC
HSC
Pluripotency
Lineage commitment
Primitive epigenetic signature
Epigenetic silencing
CMA
CMA
HSC
HSC
HSC
HPC
HPC
CMA-induced
CMA-induced
HSC expansion
HPC reprogramming back to HSC
HPC
FIGURE 1. Theoretical approaches to expand HSCs. HSCs can divide symmetrically and generate either two HSCs (self-renewal) or two HPCs (differentiation). Alternatively, HSCs can divide asymmetrically and generate one HSC and one HPC. Symmetrical renewal divisions lead to the expansion of the HSC pool, whereas asymmetrical HSC division ensures the maintenance of the HSC pool while providing a reservoir of committed HPCs and symmetrical differentiation division generates a pool of committed HPCs. A complex regulatory circuit relying on microenvironmental signals, functional and structural chromatin changes, and transcriptional regulators fine tune the HSC fate decision to maintain the proper balance between HSC expansion and differentiation. CMAs may increase the HSC numbers by promoting their self-renewal and by reprogramming HPCs to HSCs. CMA, chromatin-modifying agent; HPC, hematopoietic progenitor cell; HSC, hematopoietic stem cell.
upregulating pluripotency genes that would lead to increased numbers of HSCs (Fig. 1). The pluripotency gene-expression patterns achieved would have to be solely expressed during the period of HSC division and imprint an HSC epigenetic pattern that is inheritable but not associated with malignant transformation.
STRATEGIES TO EXPAND AND REPROGRAM HEMATOPOIETIC STEM CELLS USING EPIGENETIC MODIFIERS For several decades, drugs capable of affecting the chromatin structure including histone deacetylase inhibitors (HDACIs) and DNA methyltransferase inhibitors (DNMTIs) have been used successfully to treat patients with myelodysplastic disorders and forms of refractory acute myeloid leukemia [35]. These agents have been thought to act by inducing apoptosis and differentiation of cells belonging to the malignant clone. These same drugs appear, however, to have a far different effect on normal HSCs by promoting the generation of HPCs and HSCs from cultured normal CD34þ cells [19,24 ,25,36]. These observations raised the &&
possibility that the beneficial effects of a variety of CMAs observed in patients with myeloid malignancies might be not only because of their effects on malignant cells, but also because of their ability to reawaken the reservoir of normal HSCs that persist within such patients providing them with a competitive advantage. DNA methylation maintains persistent cellular memories and is thought to be the primary epigenetic barrier to reprogramming [11,17]. The reprogramming process affected by the enforced expression of embryonic transcription factors results in the creation of iPSCs by activating endogenous pluripotency genes including Oct4 and Nanog by influencing the methylation status of their promoter regions. These efforts can lead at times to limited activation of pluripotency genes and partially or transiently reprogrammed iPSCs [11,20 ,37]. The inclusion of DNMTIs and HDACIs has been reported to improve the reprogramming efficiency of somatic cells. The HDACIs, valproic acid (VPA) but not trichostatin or the DNMTi azacytidine can improve the efficiency of creating iPSCs by reactivating the Oct 4 promoter [25]. Several groups have hypothesized that short-term exposure
1065-6251 Copyright ß 2015 Wolters Kluwer Health, Inc. All rights reserved.
&&
www.co-hematology.com
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
281
Hematopoiesis
of primary human or murine HSCs to low doses of CMAs might also be of use for reprogramming cells generated during the ex-vivo expansion of primary HSCs [31,32]. These small molecules would presumably act by transiently reactivating genetic programs required for HSC self-renewal. The goal of such efforts is not to create immortalized cell lines that are irreversibly altered, but rather to establish an epigenetic signature for a limited but sufficient period of time so as to allow additional HSCs to be generated during a brief period of culture. Such epigenetic modifications created would be likely inheritable. The elimination of the CMAs after the period of culture would be anticipated to silence the genetic program that led to HSC expansion, thereby minimizing the risks of leukemic transformation but not compromising the genetic and functional properties of the HSCs that have already been produced. In order to explore whether CMAs were capable of breaking the barrier favoring differentiation, Milhem et al. [25] created a cytokine milieu which favored differentiation of human adult marrow CD34þ cells and compared the fate of CD34þ cells which were cultured in the presence of cytokines alone or the same cytokines accompanied by sequential treatment with decitabine followed by trichostatin A. The cells were initially cultured in a cytokine combination consisting of stem cell factor, Fms-like tyrosine kinase (FLT-3) ligand, thrombopoietin, and interleukin (IL)-3 in order to promote HSC cycling which was anticipated to be required for the incorporation of decitabine. After 16 h, the cells were exposed to low doses of decitabine and after an additional 48 h the cells were washed and exposed either to differentiating culture conditions including cytokines (stem cell factor, granulocyte– macrophage colony-stimulatng factor IL-3, IL-6, and erythropoietin) and 30% fetal bovine serum or in the presence of trichostatin A. The cells exposed to cytokines alone experienced a massive expansion of total cell numbers but a decline in the numbers of CD34þ and CD34þCD90þ cells, whereas those cells exposed to the trichostatin A underwent far less proliferation but a six-fold expansion in CD34þ cells and a 2.5-fold expansion in CD34þCD90þ cells [25]. Furthermore, cells exposed to decitabine followed by trichostatin A as well as cells exposed to decitabine alone but not cells exposed to cytokines or trichostatin A alone were capable of multilineage human engraftment in immune-deficient mice. These findings indicate that the wave of differentiation induced by cytokines can be reversed by cellular reprogramming with CMAs [25]. Similarly, Walasek et al. [31] have shown that treatment with VPA alone or in combination with lithium chloride was able to preserve 282
www.co-hematology.com
HSC function in in-vitro culture conditions which favored hematopoietic cell differentiation. They provided evidence that this preservation of HSC function was associated with upregulation of genes associated with stem cell maintenance and downregulation of genes associated with differentiation, thereby effectively negating the effects of differentiation-inducing cytokines [31]. The use of each of these CMAs was associated with transient increases of histone acetylation and DNA demethylation of genes that are frequently implicated in the expansion and maintenance of fully functional HSCs but are silenced during culturing of HSCs. The constituents of the culture media that is used for altering HSC fate decisions have an important effect on the success of cellular reprogramming. Serum contains numerous proteins which can vary from lot to lot and can affect the gene expression of cells that are being cultured in an unpredictable manner. Components of serum likely alter the gene-expression patterns by affecting the regulatory pathways favoring the induction of genes favoring differentiation and silencing the genes associated with intact HSC function [19,20 ]. These observations likely provide an explanation for the well known difference of individual lots of serum to favor the proliferation of cells belonging to particular lineage. Chaurasia et al. [19,20 ] have demonstrated in fact that the inclusion of serum in cultures of UCB CD34þ cells favors differentiation rather than an incremental increase in HSC numbers. The use of serum in culture systems that are constructed to favor cellular reprogramming, therefore, appears unwise. Different HDACIs under identical culture have different effects on HSC fate decisions [19]. Furthermore, the ability of these agents to reprogram HSCs is also affected by the source of HSCs with the greatest success being observed in the following order: UCB > bone marrow > mobilized peripheral blood CD34þ cells [26 ]. Such ontogeny-related responses are likely the result of the depth of epigenetic modifications of the expression of genes that lead to symmetrical HSC divisions and reprogramming present in these HSC sources. Such observations should be taken into account when selecting the optimal source of HSCs and CMAs to be used for these purposes. Our laboratory has recently reported that the effect of VPA on HSC expansion under serumfree conditions is related to the upregulation of a number of pluripotency genes which can be eliminated by forced downregulation of these same genes [20 ]. These findings do not preclude the possibility that other CMAs can affect HSC decisions by influencing the alternative regulatory networks. &&
&&
&
&&
Volume 22 Number 4 July 2015
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Role of epigenetic reprogramming in hsc Iancu-Rubin and Hoffman
PC
% Human cell engraftment in quaternary NSG mice
8
Control
VPA
6
* 4
* 2
* *
*
*
C D 4 C 5 D 33 C C D3 D 41 C a D 1 G 9 PA C D 4 C 5 D 33 C C D3 D 41 C a D 1 G 9 PA C D 4 C 5 D 33 C C D3 D 41 C a D 1 G 9 PA
0
FIGURE 2. VPA-expanded UCB HSCs are capable of longterm multilineage human engraftment in quaternary NSG mice. 2 106 BM cells from the tertiary recipients of unmanipulated human CD34þ cells or HSC grafts expanded in the absence or in the presence of VPA were transplanted into the quaternary NSG mice. Each bar represents the median percentage of human cell engraftment that occurred 16–17 weeks after transplantation in the marrow of quaternary recipient animals as determined by the phenotypical characterization of multilineage human hematopoietic cells (i.e. CD45, CD33, CD3, CD41, CD19, and GPA). Statistically significant. BM, bone marrow; HSCs, hematopoietic stem cells; NSG, NOD SCID gamma; PC, primary CD34þ cells; UCB, umbilical cord blood; VPA, valproic acid. &&
Furthermore, Chaurasia et al. [20 ] demonstrated that cytokines and VPA affect different processes each contributing to HSC expansion. For instance,
priming of the UCB CD34þ cells was required for HSC expansion to occur. After this priming period, the addition of VPA to serum-free media without cytokines resulted in a significant expansion of primitive CD34þC90þCD184þCD49fþCD45A- HSCs, compared with the primary UCB CD34þ cells. Importantly, these VPA-expanded HSCs retained their ability to engraft immune-compromised mice, albeit to a far lesser degree to those CD34þ cells that were exposed to VPA in the presence of continued cytokine exposure. These data indicate that the expansion of HSCs is a cumulative effect resulting from the reprogramming action of VPA and the proliferationpromoting role of cytokines in the absence of serum. HSCs expanded in this fashion were capable of multilineage engraftment in primary, secondary, tertiary as well as quaternary murine recipients (Fig. 2). The demonstration of the inability of these reprogrammed HSCs to generate teratomas or hematological malignancies in recipient mice is likely because of the transient upregulation of the pluripotency genes which was no longer observed within human cells present following serial transplantation. As HSC engraftment within the allogeneic hosts is likely because of the contributions of cells within various subpopulations within the HSC hierarchy which are responsible for rapid, short-term and long-term engraftment, one must be assured that adeqate numbers of these various HSC subpopulations are present in an expanded cell product. Such HSC subpopulations can be identified by flow cytometric analysis of CD34 cells immunolabeled
Control
VPA
ST-RC
ST-RC
CD34+
CD34+ CD90– CD49f–
CD90– CD49f–
I-RC LT-RC
LT-RC CD34+ CD90+ CD49f+
I-RC CD34+ CD90+ CD49f–
CD34+ CD90+ CD49f+
CD34+ CD90+ CD49f–
FIGURE 3. VPA-expanded UCB HSC grafts comprise hierarchical HSC subpopulations responsible for rapid, intermediate and short-term engraftment. Phenotypical characterization of VPA-expanded HSC graft by flow cytometric analysis of CD34, CD90 and CD49f expression revealed the presence of three different HSC subpopulations: CD34þCD90þCD49fþ which have LT-RC, CD34þCD90þCD49f which have I-RC, and CD34þCD90CD49f which have ST-RC. Schematic illustration of the proportion of each LT-RC, I-RC, and ST-RC subpopulations with HSCs expanded in the absence (control) or in the presence of VPA. HSC, hematopoietic stem cell; I-RC, intermediate repopulating capacity; LT-RC, long-term repopulating capacity; ST-RC, short-term repopulating capacity; UCB, umbilical cord blood; VPA, valproic acid. 1065-6251 Copyright ß 2015 Wolters Kluwer Health, Inc. All rights reserved.
www.co-hematology.com
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
283
Hematopoiesis
with monoclonal antibodies against a variety of stem cell markers, including CD38, CD90, c-kit (CD117), integrin a6 (CD49f), and CXCR4 (CD184) [38]. Treatment of UCB CD34þ with VPA under serum-free conditions have been shown to lead to the generation of greater numbers of shortterm, intermediate, and long-term repopulating cells (Fig. 3). Mahmud et al. [24 ], however, have reported under serum-containing culture system that VPA was more effective than decitabine and trichostatin A at generating more differentiated HSC populations. The reasons for these conflicting results are likely because of the use of serum in one of these reports and the cellular toxicity of decitabine when used in serum-free culture systems. &&
CONCLUSION The use of ex-vivo culture systems to expand the number of transplantable HSCs has resulted in HSC depletion and the generation of large numbers of progenitor cells as well as short-term but not longterm repopulating cells. To achieve ex-vivo HSC expansion within a limited period of time would require a technology capable of inducing not only symmetrical HSC self-renewal divisions, but also cellular reprogramming of HPCs. The use of early-acting cytokines and CMAs in serum-free culture media provides a promising strategy to achieve these goals. Acknowledgements None. Financial support and sponsorship This work is supported by the Empire State Stem Cell Board; NYSTEM Program. Conflicts of interest There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as: & of special interest && of outstanding interest 1. Dahlberg A, Delaney C, Bernstein ID. Ex vivo expansion of human hematopoietic stem and progenitor cells. Blood 2011; 117:6083–6090. 2. Wagner JE Jr, Eapen M, Carter S, et al. One-unit versus two-unit cord-blood & transplantation for hematologic cancers. N Engl J Med 2014; 371:1685– 1694. A surprising clinical trial indicating limitations of double cord-blood transplant. 3. Boitano AE, Wang J, Romeo R, et al. Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells. Science 2010; 329:1345–1348. 4. De Lima M, McNiece I, Robinson SN, et al. Cord-blood engraftment with ex vivo mesenchymal-cell coculture. N Engl J Med 2012; 367:2305–2315. 5. Delaney C, Ratajczak MZ, Laughlin MJ. Strategies to enhance umbilical cord blood stem cell engraftment in adult patients. Expert Rev Hematol 2010; 3:273–283.
284
www.co-hematology.com
6. Himburg HA, Muramoto GG, Daher P, et al. Pleiotrophin regulates the expansion and regeneration of hematopoietic stem cells. Nat Med 2010; 16:475–482. 7. Nishino T, Miyaji K, Ishiwata N, et al. Ex vivo expansion of human hematopoietic stem cells by a small-molecule agonist of c-MPL. Exp Hematol 2009; 37:1364.e4–1377.e4. 8. North TE, Goessling W, Walkley CR, et al. Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis. Nature 2007; 447:1007–1011. 9. Walasek MA, van Os R, de Haan G. Hematopoietic stem cell expansion: challenges and opportunities. Ann N Y Acad Sci 2012; 1266:138–150. 10. Fares I, Chagraoui J, Gareau Y, et al. Cord blood expansion. Pyrimidoindole derivatives are agonists of human hematopoietic stem cell self-renewal. Science 2014; 345:1509–1512. 11. Apostolou E, Hochedlinger K. Chromatin dynamics during cellular reprogramming. Nature 2013; 502:462–471. 12. Cabezas-Wallscheid N, Klimmeck D, Hansson J, et al. Identification of && regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis. Cell Stem Cell 2014; 15:507–522. A comprehensive study describing the epigenetic regulatory networks in HSCs. 13. DeVilbiss AW, Sanalkumar R, Johnson KD, et al. Hematopoietic transcriptional mechanisms: from locus-specific to genome-wide vantage points. Exp Hematol 2014; 42:618–629. 14. Hochedlinger K, Plath K. Epigenetic reprogramming and induced pluripotency. Development 2009; 136:509–523. 15. Huang HT, Kathrein KL, Barton A, et al. A network of epigenetic regulators guides developmental haematopoiesis in vivo. Nat Cell Biol 2013; 15:1516– 1525. 16. Schonheit J, Leutz A, Rosenbauer F. Chromatin dynamics during differentiation of myeloid cells. J Mol Biol 2015; 427:670–687. 17. Watanabe A, Yamada Y, Yamanaka S. Epigenetic regulation in pluripotent stem cells: a key to breaking the epigenetic barrier. Philos Trans R Soc Lond B Biol Sci 2013; 368:20120292. 18. Bug G, Gul H, Schwarz K, et al. Valproic acid stimulates proliferation and selfrenewal of hematopoietic stem cells. Cancer Res 2005; 65:2537–2541. 19. Chaurasia P, Berenzon D, Hoffman R. Chromatin-modifying agents promote the ex vivo production of functional human erythroid progenitor cells. Blood 2011; 117:4632–4641. 20. Chaurasia P, Gajzer DC, Schaniel C, et al. Epigenetic reprogramming induces && the expansion of cord blood stem cells. J Clin Invest 2014; 124:2378–2395. A technical report on the use of valproic acid to expand human HSCs. Expansion of HSCs was associated with the activation of pluripotency genes. 21. De Felice L, Tatarelli C, Mascolo MG, et al. Histone deacetylase inhibitor valproic acid enhances the cytokine-induced expansion of human hematopoietic stem cells. Cancer Res 2005; 65:1505–1513. 22. Kaur K, Mirlashari MR, Kvalheim G, Kjeldsen-Kragh J. 30 ,40 -Dimethoxyflavone and valproic acid promotes the proliferation of human hematopoietic stem cells. Stem Cell Res Ther 2013; 4:60–68. 23. Liu J, Samuel K, Turner ML, Gallagher RC. Use of IL3 and chromatin-modifying reagents valproic acid and 5-aza-20 -deoxycytidine to affect mobilized peripheral blood CD34þ cell fate decisions. Vox Sang 2014; 107:83–89. 24. Mahmud N, Petro B, Baluchamy S, et al. Differential effects of epigenetic && modifiers on the expansion and maintenance of human cord blood stem/ progenitor cells. Biol Blood Marrow Transplant 2014; 20:480–489. An excellent paper describing the possibility that various CMAs have different ability to expand progenitor and stem cells. 25. Milhem M, Mahmud N, Lavelle D, et al. Modification of hematopoietic stem cell fate by 5aza 20 deoxycytidine and trichostatin A. Blood 2004; 103:4102– 4110. 26. Saraf S, Araki H, Petro B, et al. Ex vivo expansion of human mobilized & peripheral blood stem cells using epigenetic modifiers. Transfusion 2014; doi: 10.1111/trf.12904. [Epub ahead of print] Important study showing that CMAs have different effects on different sources of human hematopoietic cells. 27. Seet LF, Teng E, Lai YS, et al. Valproic acid enhances the engraftability of human umbilical cord blood hematopoietic stem cells expanded under serumfree conditions. Eur J Haematol 2009; 82:124–132. 28. Teng HF, Li PN, Hou DR, et al. Valproic acid enhances Oct4 promoter activity through PI3K/Akt/mTOR pathway activated nuclear receptors. Mol Cell Endocrinol 2014; 383:147–158. 29. Trecul A, Morceau F, Gaigneaux A, et al. Valproic acid regulates erythromegakaryocytic differentiation through the modulation of transcription factors and microRNA regulatory micro-networks. Biochem Pharmacol 2014; 92:299–311. 30. Vulcano F, Milazzo L, Ciccarelli C, et al. Valproic acid affects the engraftment of TPO-expanded cord blood cells in NOD/SCID mice. Exp Cell Res 2012; 318:400–407. 31. Walasek MA, Bystrykh L, van den Boom V, et al. The combination of valproic acid and lithium delays hematopoietic stem/progenitor cell differentiation. Blood 2012; 119:3050–3059. 32. Walasek MA, Bystrykh LV, Olthof S, et al. Sca-1 is an early-response target of histone deacetylase inhibitors and marks hematopoietic cells with enhanced function. Exp Hematol 2013; 41:113.e2 –123.e2.
Volume 22 Number 4 July 2015
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Role of epigenetic reprogramming in hsc Iancu-Rubin and Hoffman 33. Zini R, Norfo R, Ferrari F, et al. Valproic acid triggers erythro/megakaryocyte lineage decision through induction of GFI1B and MLLT3 expression. Exp Hematol 2012; 40:1043.e6–1054.e6. 34. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126:663–676. 35. Santini V. Life after hypomethylating agents in myelodysplastic syndrome: new strategies. Curr Opin Hematol 2015; 22:155–162.
36. Broxmeyer HE. Inhibiting HDAC for human hematopoietic stem cell expansion. J Clin Invest 2014; 124:2365–2368. 37. Kang SJ, Park YI, So B, Kang HG. Sodium butyrate efficiently converts fully reprogrammed induced pluripotent stem cells from mouse partially reprogrammed cells. Cell Reprogram 2014; 16:345–354. 38. Notta F, Doulatov S, Laurenti E, et al. Isolation of single human hematopoietic stem cells capable of long-term multilineage engraftment. Science 2011; 333:218–221.
1065-6251 Copyright ß 2015 Wolters Kluwer Health, Inc. All rights reserved.
www.co-hematology.com
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
285
Role of epigenetic reprogramming in hematopoietic stem cell function Camelia Iancu-Rubin and Ronald Hoffman
Purpose of review Epigenetic regulatory networks determine the fate of dividing hematopoietic stem cells (HSCs). Prior attempts at the ex-vivo expansion of transplantable human HSCs have led to the depletion or at best maintenance of the numbers of HSCs because of the epigenetic events that silence the HSC geneexpression pattern. The purpose of this review is to outline the recent efforts to use small molecules to reprogram cultured CD34þ cells so as to expand their numbers. Recent findings Chromatin-modifying agents (CMAs) reactivate the gene-expression patterns of HSCs that have been silenced as they divide ex vivo. Increasing evidence indicates that CMAs act not only by promoting HSC symmetrical self-renewal divisions, but also by reprogramming progenitor cells, resulting in greater numbers of HSCs. The use of such CMAs for these purposes has not resulted in malignant transformation of the ex-vivo treated cell product. Summary The silencing of the gene-expression program that determines HSC function after ex-vivo culture can be reversed by reprogramming the progeny of dividing HSCs with transient exposure to CMAs. The successful implementation of this approach provides a strategy which might lead to the development of a clinically relevant means of manufacturing increased numbers of HSCs. Keywords epigenetic reprogramming, hematopoietic stem cell expansion, hematopoietic stem cell transplantation
INTRODUCTION
single UCB graft did not lead to a survival advantage in either children or adolescents [2 ]. These observations have led to renewed interest in creating exvivo strategies to expand the number of HSCs within a single UCB collection. Such efforts are not only of clinical importance, but also would lead to a greater understanding of the events that determine the fate of dividing HSCs. &
Hematopoietic stem cell (HSC) transplantation provides patients with refractory hematological malignancies or genetic disorders due to blood cell abnormalities an opportunity for cure. Lack of access to a matched histocompatible allogeneic sibling, unrelated matched HSC donor or haploidentical donor prevents many such patients from receiving such therapy [1]. The identification of unrelated umbilical cord blood (UCB) as a readily available source of such normal HSC grafts has provided another alternative HSC source. As there are a fixed, limited number of HSCs within a single UCB collection, the clinical outcomes of adults receiving such grafts have been less favorable than that observed in children receiving UCB collections [1]. Such limitations have resulted in the infusion of two different matched UCB collections in order to increase the number of HSCs. Although several phase 2 trials have provided promising results, a more extensive phase 3 trial concluded that transplantation with two UCB units compared with a
PRIOR ATTEMPTS TO EXPAND HEMATOPOIETIC STEM CELLS The initial efforts to expand HSCs ex vivo attempted to recreate a hematopoietic marrow microenvironment The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA Correspondence to Ronald Hoffman, MD, Division of Hematology and Medical Oncology, Box 1079, 1 Gustave L. Levy Place, New York, NY 10029, USA. Tel: +1 212 241 2296; e-mail: [email protected] Curr Opin Hematol 2015, 22:279–285 DOI:10.1097/MOH.0000000000000143
1065-6251 Copyright ß 2015 Wolters Kluwer Health, Inc. All rights reserved.
www.co-hematology.com
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Hematopoiesis
KEY POINTS Epigenetic regulation is a key factor determining the HSC fate decision. Chromatin-modifying agents can promote HSC expansion by inducing HSC symmetrical self-renewal and by reprogramming HPC to HSC. HSC grafts expanded by means of epigenetic reprogramming are a potential source for allogeneic HSC transplantation for patients with refractory hematological malignancies or genetic blood disorders.
which favored symmetrical stem cell expansion [3– 9]. For these purposes, combinations of cytokines known to be elaborated by marrow microenvironmental cells and to promote HSC cycling and their subsequent division were added. Such efforts, however, resulted in the generation of large numbers of hematopoietic progenitor cells (HPCs) and precursor cells but reduced numbers of HSCs. The rapid ex-vivo cycling and division of cord blood CD34þ cells that occurred in the presence of such cytokine combinations led to HSC commitment, with the residual marrow-repopulating potential being attributed to a small fraction of HSCs that remained quiescent or had undergone a limited number of cell divisions. Mesenchymal cell feeder layers or a number of molecules such as immobilized notch ligand, a copper chelator, an aryl hydrocarbon receptor antagonist (SR1), prostaglandin E2 (PGE2), and a pyrimidoindole derivative (UM171) have been added to such cytokine combinations with the hope of further expanding the number of transplantable cord blood HSCs [3–8,10]. To date, these attempts have met with limited success toward clinical applications. The reconstruction of a marrow microenvironment that favors stem HSC expansion ex vivo appears to be an elusive goal as the components of the niche which favor HSC self-renewal remain uncertain and the ability of bioengineers to construct such a dynamic and complex environment which would be sustainable ex vivo for prolonged periods of time would require overcoming numerous obstacles.
STEM CELL DECISIONS AND EPIGENETIC MODIFICATIONS HSCs are able to balance the self-renewal with commitment in vivo by controlling the proportion of asymmetric and symmetric cell divisions that they undergo, but this balance is lost following ex-vivo culture (Fig. 1). During asymmetric HSC division, one daughter cell remains an HSC identical to the mother cell, whereas the other becomes committed 280
www.co-hematology.com
and develops into a HPC. By contrast, during symmetrical division a HSC can divide to generate either two HSCs (symmetrical renewal) or two committed HPCs (symmetrical commitment). Asymmetrical HSC division results in the maintenance of the numbers of HSCs while allowing for a progressive increase in the numbers of HPCs. Symmetrical renewal divisions, by contrast, lead to an expansion of the pool of HSCs, whereas symmetrical commitment divisions result in the generation of differentiated cells and eventual HSC exhaustion. A growing number of investigators have speculated that dynamic epigenetic events alter the chromatin structure and lead to transcriptional programs that determine the HSC status [11,12 ,13–17]. In vivo, these epigenetic events likely occur in response to a variety of microenvironmental cues which change during development or occur in response to external stimuli or demands requiring alterations in blood cell production. Gene expression is dynamically regulated by the modifications of chromatin which occurs largely as a consequence of DNA methylation and histone acetylation [12 ]. Several groups, including our own, have used chromatin-modifying agents (CMAs) to treat HSCs in vitro in order to favor the expression of genetic programs which support symmetrical renewal divisions or to reprogram primitive HPCs back to HSCs [18,19,20 ,21–23,24 ,25,26 , 27–33]. Ex-vivo culture conditions represent stressful conditions which frequently lead to HSC commitment. As HSCs are slow cycling cells, a prolonged period of culture would be needed if the expansion of HSCs as entirely dependent on a significant fraction of HSCs to undergo symmetrical self-renewing divisions. The period of ex-vivo treatment could be theoretically shortened if the agent or agents used to expand HSCs were also capable of reprogramming more differentiated HPCs. Cellular reprogramming induces differentiated cells to revert back to undifferentiated cells. The epigenetic barrier for reprogramming mammalian cells has been overcome with the use of reprogramming factors such a Oct3, Oct4, Sox2, Klf4, Myc, and Lin28 [14]. Such an approach was utilized by Takahashi and Yamanaka [34] to create immortalized pluripotent stem cells (iPSCs). This approach, however, is not currently suitable to generate larger numbers of transplantable HSCs because of the safety concerns associated with their propensity to undergo maligant transformation (i.e. formation of teratomas) and the limited engaftment potential of iPSC-derived cells with an HSC phenotype. For the strategy of reprogramming of UCB HSCs to be clinically applicable, it should be not only capable of promoting the division of primitive HSC populations, but also capable of transiently &&
&&
&&
&&
&
Volume 22 Number 4 July 2015
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Role of epigenetic reprogramming in hsc Iancu-Rubin and Hoffman
Symmetrical
Asymmetrical
HSC
Symmetrical
HSC
HSC
Pluripotency
Lineage commitment
Primitive epigenetic signature
Epigenetic silencing
CMA
CMA
HSC
HSC
HSC
HPC
HPC
CMA-induced
CMA-induced
HSC expansion
HPC reprogramming back to HSC
HPC
FIGURE 1. Theoretical approaches to expand HSCs. HSCs can divide symmetrically and generate either two HSCs (self-renewal) or two HPCs (differentiation). Alternatively, HSCs can divide asymmetrically and generate one HSC and one HPC. Symmetrical renewal divisions lead to the expansion of the HSC pool, whereas asymmetrical HSC division ensures the maintenance of the HSC pool while providing a reservoir of committed HPCs and symmetrical differentiation division generates a pool of committed HPCs. A complex regulatory circuit relying on microenvironmental signals, functional and structural chromatin changes, and transcriptional regulators fine tune the HSC fate decision to maintain the proper balance between HSC expansion and differentiation. CMAs may increase the HSC numbers by promoting their self-renewal and by reprogramming HPCs to HSCs. CMA, chromatin-modifying agent; HPC, hematopoietic progenitor cell; HSC, hematopoietic stem cell.
upregulating pluripotency genes that would lead to increased numbers of HSCs (Fig. 1). The pluripotency gene-expression patterns achieved would have to be solely expressed during the period of HSC division and imprint an HSC epigenetic pattern that is inheritable but not associated with malignant transformation.
STRATEGIES TO EXPAND AND REPROGRAM HEMATOPOIETIC STEM CELLS USING EPIGENETIC MODIFIERS For several decades, drugs capable of affecting the chromatin structure including histone deacetylase inhibitors (HDACIs) and DNA methyltransferase inhibitors (DNMTIs) have been used successfully to treat patients with myelodysplastic disorders and forms of refractory acute myeloid leukemia [35]. These agents have been thought to act by inducing apoptosis and differentiation of cells belonging to the malignant clone. These same drugs appear, however, to have a far different effect on normal HSCs by promoting the generation of HPCs and HSCs from cultured normal CD34þ cells [19,24 ,25,36]. These observations raised the &&
possibility that the beneficial effects of a variety of CMAs observed in patients with myeloid malignancies might be not only because of their effects on malignant cells, but also because of their ability to reawaken the reservoir of normal HSCs that persist within such patients providing them with a competitive advantage. DNA methylation maintains persistent cellular memories and is thought to be the primary epigenetic barrier to reprogramming [11,17]. The reprogramming process affected by the enforced expression of embryonic transcription factors results in the creation of iPSCs by activating endogenous pluripotency genes including Oct4 and Nanog by influencing the methylation status of their promoter regions. These efforts can lead at times to limited activation of pluripotency genes and partially or transiently reprogrammed iPSCs [11,20 ,37]. The inclusion of DNMTIs and HDACIs has been reported to improve the reprogramming efficiency of somatic cells. The HDACIs, valproic acid (VPA) but not trichostatin or the DNMTi azacytidine can improve the efficiency of creating iPSCs by reactivating the Oct 4 promoter [25]. Several groups have hypothesized that short-term exposure
1065-6251 Copyright ß 2015 Wolters Kluwer Health, Inc. All rights reserved.
&&
www.co-hematology.com
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
281
Hematopoiesis
of primary human or murine HSCs to low doses of CMAs might also be of use for reprogramming cells generated during the ex-vivo expansion of primary HSCs [31,32]. These small molecules would presumably act by transiently reactivating genetic programs required for HSC self-renewal. The goal of such efforts is not to create immortalized cell lines that are irreversibly altered, but rather to establish an epigenetic signature for a limited but sufficient period of time so as to allow additional HSCs to be generated during a brief period of culture. Such epigenetic modifications created would be likely inheritable. The elimination of the CMAs after the period of culture would be anticipated to silence the genetic program that led to HSC expansion, thereby minimizing the risks of leukemic transformation but not compromising the genetic and functional properties of the HSCs that have already been produced. In order to explore whether CMAs were capable of breaking the barrier favoring differentiation, Milhem et al. [25] created a cytokine milieu which favored differentiation of human adult marrow CD34þ cells and compared the fate of CD34þ cells which were cultured in the presence of cytokines alone or the same cytokines accompanied by sequential treatment with decitabine followed by trichostatin A. The cells were initially cultured in a cytokine combination consisting of stem cell factor, Fms-like tyrosine kinase (FLT-3) ligand, thrombopoietin, and interleukin (IL)-3 in order to promote HSC cycling which was anticipated to be required for the incorporation of decitabine. After 16 h, the cells were exposed to low doses of decitabine and after an additional 48 h the cells were washed and exposed either to differentiating culture conditions including cytokines (stem cell factor, granulocyte– macrophage colony-stimulatng factor IL-3, IL-6, and erythropoietin) and 30% fetal bovine serum or in the presence of trichostatin A. The cells exposed to cytokines alone experienced a massive expansion of total cell numbers but a decline in the numbers of CD34þ and CD34þCD90þ cells, whereas those cells exposed to the trichostatin A underwent far less proliferation but a six-fold expansion in CD34þ cells and a 2.5-fold expansion in CD34þCD90þ cells [25]. Furthermore, cells exposed to decitabine followed by trichostatin A as well as cells exposed to decitabine alone but not cells exposed to cytokines or trichostatin A alone were capable of multilineage human engraftment in immune-deficient mice. These findings indicate that the wave of differentiation induced by cytokines can be reversed by cellular reprogramming with CMAs [25]. Similarly, Walasek et al. [31] have shown that treatment with VPA alone or in combination with lithium chloride was able to preserve 282
www.co-hematology.com
HSC function in in-vitro culture conditions which favored hematopoietic cell differentiation. They provided evidence that this preservation of HSC function was associated with upregulation of genes associated with stem cell maintenance and downregulation of genes associated with differentiation, thereby effectively negating the effects of differentiation-inducing cytokines [31]. The use of each of these CMAs was associated with transient increases of histone acetylation and DNA demethylation of genes that are frequently implicated in the expansion and maintenance of fully functional HSCs but are silenced during culturing of HSCs. The constituents of the culture media that is used for altering HSC fate decisions have an important effect on the success of cellular reprogramming. Serum contains numerous proteins which can vary from lot to lot and can affect the gene expression of cells that are being cultured in an unpredictable manner. Components of serum likely alter the gene-expression patterns by affecting the regulatory pathways favoring the induction of genes favoring differentiation and silencing the genes associated with intact HSC function [19,20 ]. These observations likely provide an explanation for the well known difference of individual lots of serum to favor the proliferation of cells belonging to particular lineage. Chaurasia et al. [19,20 ] have demonstrated in fact that the inclusion of serum in cultures of UCB CD34þ cells favors differentiation rather than an incremental increase in HSC numbers. The use of serum in culture systems that are constructed to favor cellular reprogramming, therefore, appears unwise. Different HDACIs under identical culture have different effects on HSC fate decisions [19]. Furthermore, the ability of these agents to reprogram HSCs is also affected by the source of HSCs with the greatest success being observed in the following order: UCB > bone marrow > mobilized peripheral blood CD34þ cells [26 ]. Such ontogeny-related responses are likely the result of the depth of epigenetic modifications of the expression of genes that lead to symmetrical HSC divisions and reprogramming present in these HSC sources. Such observations should be taken into account when selecting the optimal source of HSCs and CMAs to be used for these purposes. Our laboratory has recently reported that the effect of VPA on HSC expansion under serumfree conditions is related to the upregulation of a number of pluripotency genes which can be eliminated by forced downregulation of these same genes [20 ]. These findings do not preclude the possibility that other CMAs can affect HSC decisions by influencing the alternative regulatory networks. &&
&&
&
&&
Volume 22 Number 4 July 2015
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Role of epigenetic reprogramming in hsc Iancu-Rubin and Hoffman
PC
% Human cell engraftment in quaternary NSG mice
8
Control
VPA
6
* 4
* 2
* *
*
*
C D 4 C 5 D 33 C C D3 D 41 C a D 1 G 9 PA C D 4 C 5 D 33 C C D3 D 41 C a D 1 G 9 PA C D 4 C 5 D 33 C C D3 D 41 C a D 1 G 9 PA
0
FIGURE 2. VPA-expanded UCB HSCs are capable of longterm multilineage human engraftment in quaternary NSG mice. 2 106 BM cells from the tertiary recipients of unmanipulated human CD34þ cells or HSC grafts expanded in the absence or in the presence of VPA were transplanted into the quaternary NSG mice. Each bar represents the median percentage of human cell engraftment that occurred 16–17 weeks after transplantation in the marrow of quaternary recipient animals as determined by the phenotypical characterization of multilineage human hematopoietic cells (i.e. CD45, CD33, CD3, CD41, CD19, and GPA). Statistically significant. BM, bone marrow; HSCs, hematopoietic stem cells; NSG, NOD SCID gamma; PC, primary CD34þ cells; UCB, umbilical cord blood; VPA, valproic acid. &&
Furthermore, Chaurasia et al. [20 ] demonstrated that cytokines and VPA affect different processes each contributing to HSC expansion. For instance,
priming of the UCB CD34þ cells was required for HSC expansion to occur. After this priming period, the addition of VPA to serum-free media without cytokines resulted in a significant expansion of primitive CD34þC90þCD184þCD49fþCD45A- HSCs, compared with the primary UCB CD34þ cells. Importantly, these VPA-expanded HSCs retained their ability to engraft immune-compromised mice, albeit to a far lesser degree to those CD34þ cells that were exposed to VPA in the presence of continued cytokine exposure. These data indicate that the expansion of HSCs is a cumulative effect resulting from the reprogramming action of VPA and the proliferationpromoting role of cytokines in the absence of serum. HSCs expanded in this fashion were capable of multilineage engraftment in primary, secondary, tertiary as well as quaternary murine recipients (Fig. 2). The demonstration of the inability of these reprogrammed HSCs to generate teratomas or hematological malignancies in recipient mice is likely because of the transient upregulation of the pluripotency genes which was no longer observed within human cells present following serial transplantation. As HSC engraftment within the allogeneic hosts is likely because of the contributions of cells within various subpopulations within the HSC hierarchy which are responsible for rapid, short-term and long-term engraftment, one must be assured that adeqate numbers of these various HSC subpopulations are present in an expanded cell product. Such HSC subpopulations can be identified by flow cytometric analysis of CD34 cells immunolabeled
Control
VPA
ST-RC
ST-RC
CD34+
CD34+ CD90– CD49f–
CD90– CD49f–
I-RC LT-RC
LT-RC CD34+ CD90+ CD49f+
I-RC CD34+ CD90+ CD49f–
CD34+ CD90+ CD49f+
CD34+ CD90+ CD49f–
FIGURE 3. VPA-expanded UCB HSC grafts comprise hierarchical HSC subpopulations responsible for rapid, intermediate and short-term engraftment. Phenotypical characterization of VPA-expanded HSC graft by flow cytometric analysis of CD34, CD90 and CD49f expression revealed the presence of three different HSC subpopulations: CD34þCD90þCD49fþ which have LT-RC, CD34þCD90þCD49f which have I-RC, and CD34þCD90CD49f which have ST-RC. Schematic illustration of the proportion of each LT-RC, I-RC, and ST-RC subpopulations with HSCs expanded in the absence (control) or in the presence of VPA. HSC, hematopoietic stem cell; I-RC, intermediate repopulating capacity; LT-RC, long-term repopulating capacity; ST-RC, short-term repopulating capacity; UCB, umbilical cord blood; VPA, valproic acid. 1065-6251 Copyright ß 2015 Wolters Kluwer Health, Inc. All rights reserved.
www.co-hematology.com
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
283
Hematopoiesis
with monoclonal antibodies against a variety of stem cell markers, including CD38, CD90, c-kit (CD117), integrin a6 (CD49f), and CXCR4 (CD184) [38]. Treatment of UCB CD34þ with VPA under serum-free conditions have been shown to lead to the generation of greater numbers of shortterm, intermediate, and long-term repopulating cells (Fig. 3). Mahmud et al. [24 ], however, have reported under serum-containing culture system that VPA was more effective than decitabine and trichostatin A at generating more differentiated HSC populations. The reasons for these conflicting results are likely because of the use of serum in one of these reports and the cellular toxicity of decitabine when used in serum-free culture systems. &&
CONCLUSION The use of ex-vivo culture systems to expand the number of transplantable HSCs has resulted in HSC depletion and the generation of large numbers of progenitor cells as well as short-term but not longterm repopulating cells. To achieve ex-vivo HSC expansion within a limited period of time would require a technology capable of inducing not only symmetrical HSC self-renewal divisions, but also cellular reprogramming of HPCs. The use of early-acting cytokines and CMAs in serum-free culture media provides a promising strategy to achieve these goals. Acknowledgements None. Financial support and sponsorship This work is supported by the Empire State Stem Cell Board; NYSTEM Program. Conflicts of interest There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as: & of special interest && of outstanding interest 1. Dahlberg A, Delaney C, Bernstein ID. Ex vivo expansion of human hematopoietic stem and progenitor cells. Blood 2011; 117:6083–6090. 2. Wagner JE Jr, Eapen M, Carter S, et al. One-unit versus two-unit cord-blood & transplantation for hematologic cancers. N Engl J Med 2014; 371:1685– 1694. A surprising clinical trial indicating limitations of double cord-blood transplant. 3. Boitano AE, Wang J, Romeo R, et al. Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells. Science 2010; 329:1345–1348. 4. De Lima M, McNiece I, Robinson SN, et al. Cord-blood engraftment with ex vivo mesenchymal-cell coculture. N Engl J Med 2012; 367:2305–2315. 5. Delaney C, Ratajczak MZ, Laughlin MJ. Strategies to enhance umbilical cord blood stem cell engraftment in adult patients. Expert Rev Hematol 2010; 3:273–283.
284
www.co-hematology.com
6. Himburg HA, Muramoto GG, Daher P, et al. Pleiotrophin regulates the expansion and regeneration of hematopoietic stem cells. Nat Med 2010; 16:475–482. 7. Nishino T, Miyaji K, Ishiwata N, et al. Ex vivo expansion of human hematopoietic stem cells by a small-molecule agonist of c-MPL. Exp Hematol 2009; 37:1364.e4–1377.e4. 8. North TE, Goessling W, Walkley CR, et al. Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis. Nature 2007; 447:1007–1011. 9. Walasek MA, van Os R, de Haan G. Hematopoietic stem cell expansion: challenges and opportunities. Ann N Y Acad Sci 2012; 1266:138–150. 10. Fares I, Chagraoui J, Gareau Y, et al. Cord blood expansion. Pyrimidoindole derivatives are agonists of human hematopoietic stem cell self-renewal. Science 2014; 345:1509–1512. 11. Apostolou E, Hochedlinger K. Chromatin dynamics during cellular reprogramming. Nature 2013; 502:462–471. 12. Cabezas-Wallscheid N, Klimmeck D, Hansson J, et al. Identification of && regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis. Cell Stem Cell 2014; 15:507–522. A comprehensive study describing the epigenetic regulatory networks in HSCs. 13. DeVilbiss AW, Sanalkumar R, Johnson KD, et al. Hematopoietic transcriptional mechanisms: from locus-specific to genome-wide vantage points. Exp Hematol 2014; 42:618–629. 14. Hochedlinger K, Plath K. Epigenetic reprogramming and induced pluripotency. Development 2009; 136:509–523. 15. Huang HT, Kathrein KL, Barton A, et al. A network of epigenetic regulators guides developmental haematopoiesis in vivo. Nat Cell Biol 2013; 15:1516– 1525. 16. Schonheit J, Leutz A, Rosenbauer F. Chromatin dynamics during differentiation of myeloid cells. J Mol Biol 2015; 427:670–687. 17. Watanabe A, Yamada Y, Yamanaka S. Epigenetic regulation in pluripotent stem cells: a key to breaking the epigenetic barrier. Philos Trans R Soc Lond B Biol Sci 2013; 368:20120292. 18. Bug G, Gul H, Schwarz K, et al. Valproic acid stimulates proliferation and selfrenewal of hematopoietic stem cells. Cancer Res 2005; 65:2537–2541. 19. Chaurasia P, Berenzon D, Hoffman R. Chromatin-modifying agents promote the ex vivo production of functional human erythroid progenitor cells. Blood 2011; 117:4632–4641. 20. Chaurasia P, Gajzer DC, Schaniel C, et al. Epigenetic reprogramming induces && the expansion of cord blood stem cells. J Clin Invest 2014; 124:2378–2395. A technical report on the use of valproic acid to expand human HSCs. Expansion of HSCs was associated with the activation of pluripotency genes. 21. De Felice L, Tatarelli C, Mascolo MG, et al. Histone deacetylase inhibitor valproic acid enhances the cytokine-induced expansion of human hematopoietic stem cells. Cancer Res 2005; 65:1505–1513. 22. Kaur K, Mirlashari MR, Kvalheim G, Kjeldsen-Kragh J. 30 ,40 -Dimethoxyflavone and valproic acid promotes the proliferation of human hematopoietic stem cells. Stem Cell Res Ther 2013; 4:60–68. 23. Liu J, Samuel K, Turner ML, Gallagher RC. Use of IL3 and chromatin-modifying reagents valproic acid and 5-aza-20 -deoxycytidine to affect mobilized peripheral blood CD34þ cell fate decisions. Vox Sang 2014; 107:83–89. 24. Mahmud N, Petro B, Baluchamy S, et al. Differential effects of epigenetic && modifiers on the expansion and maintenance of human cord blood stem/ progenitor cells. Biol Blood Marrow Transplant 2014; 20:480–489. An excellent paper describing the possibility that various CMAs have different ability to expand progenitor and stem cells. 25. Milhem M, Mahmud N, Lavelle D, et al. Modification of hematopoietic stem cell fate by 5aza 20 deoxycytidine and trichostatin A. Blood 2004; 103:4102– 4110. 26. Saraf S, Araki H, Petro B, et al. Ex vivo expansion of human mobilized & peripheral blood stem cells using epigenetic modifiers. Transfusion 2014; doi: 10.1111/trf.12904. [Epub ahead of print] Important study showing that CMAs have different effects on different sources of human hematopoietic cells. 27. Seet LF, Teng E, Lai YS, et al. Valproic acid enhances the engraftability of human umbilical cord blood hematopoietic stem cells expanded under serumfree conditions. Eur J Haematol 2009; 82:124–132. 28. Teng HF, Li PN, Hou DR, et al. Valproic acid enhances Oct4 promoter activity through PI3K/Akt/mTOR pathway activated nuclear receptors. Mol Cell Endocrinol 2014; 383:147–158. 29. Trecul A, Morceau F, Gaigneaux A, et al. Valproic acid regulates erythromegakaryocytic differentiation through the modulation of transcription factors and microRNA regulatory micro-networks. Biochem Pharmacol 2014; 92:299–311. 30. Vulcano F, Milazzo L, Ciccarelli C, et al. Valproic acid affects the engraftment of TPO-expanded cord blood cells in NOD/SCID mice. Exp Cell Res 2012; 318:400–407. 31. Walasek MA, Bystrykh L, van den Boom V, et al. The combination of valproic acid and lithium delays hematopoietic stem/progenitor cell differentiation. Blood 2012; 119:3050–3059. 32. Walasek MA, Bystrykh LV, Olthof S, et al. Sca-1 is an early-response target of histone deacetylase inhibitors and marks hematopoietic cells with enhanced function. Exp Hematol 2013; 41:113.e2 –123.e2.
Volume 22 Number 4 July 2015
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Role of epigenetic reprogramming in hsc Iancu-Rubin and Hoffman 33. Zini R, Norfo R, Ferrari F, et al. Valproic acid triggers erythro/megakaryocyte lineage decision through induction of GFI1B and MLLT3 expression. Exp Hematol 2012; 40:1043.e6–1054.e6. 34. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126:663–676. 35. Santini V. Life after hypomethylating agents in myelodysplastic syndrome: new strategies. Curr Opin Hematol 2015; 22:155–162.
36. Broxmeyer HE. Inhibiting HDAC for human hematopoietic stem cell expansion. J Clin Invest 2014; 124:2365–2368. 37. Kang SJ, Park YI, So B, Kang HG. Sodium butyrate efficiently converts fully reprogrammed induced pluripotent stem cells from mouse partially reprogrammed cells. Cell Reprogram 2014; 16:345–354. 38. Notta F, Doulatov S, Laurenti E, et al. Isolation of single human hematopoietic stem cells capable of long-term multilineage engraftment. Science 2011; 333:218–221.
1065-6251 Copyright ß 2015 Wolters Kluwer Health, Inc. All rights reserved.
www.co-hematology.com
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
285
