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 . 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 . 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 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 ex-vivo 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.
PRIOR ATTEMPTS TO EXPAND HEMATOPOIETIC STEM CELLS
The initial efforts to expand HSCs ex vivo attempted to recreate a hematopoietic marrow microenvironment 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 CD34+ 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 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 . Such an approach was utilized by Takahashi and Yamanaka  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 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 . 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 CD34+ 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 . Several groups have hypothesized that short-term exposure 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. created a cytokine milieu which favored differentiation of human adult marrow CD34+ cells and compared the fate of CD34+ 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 CD34+ and CD34+CD90+ cells, whereas those cells exposed to the trichostatin A underwent far less proliferation but a six-fold expansion in CD34+ cells and a 2.5-fold expansion in CD34+CD90+ cells . 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 . Similarly, Walasek et al. have shown that treatment with VPA alone or in combination with lithium chloride was able to preserve 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 . 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 CD34+ 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 . 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 CD34+ 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 serum-free 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. Furthermore, Chaurasia et al.[20▪▪] demonstrated that cytokines and VPA affect different processes each contributing to HSC expansion. For instance, priming of the UCB CD34+ 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 CD34+C90+CD184+CD49f+CD45A- HSCs, compared with the primary UCB CD34+ cells. Importantly, these VPA-expanded HSCs retained their ability to engraft immune-compromised mice, albeit to a far lesser degree to those CD34+ 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 proliferation-promoting 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 with monoclonal antibodies against a variety of stem cell markers, including CD38, CD90, c-kit (CD117), integrin α6 (CD49f), and CXCR4 (CD184) . Treatment of UCB CD34+ with VPA under serum-free conditions have been shown to lead to the generation of greater numbers of short-term, 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.
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 long-term 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.
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
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