- Email: [email protected]
Dramatic Expansion of HSCs: New Possibilities for HSC Transplants?
Cell Stem Cell
In Translation Dramatic Expansion of HSCs: New Possibilities for HSC Transplants? Sarah Nikiforow1,2 and Jerome Ritz1,2,* 1Division
of Hematologic Malignancies, Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA Medical School, Boston, MA 02115, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.stem.2015.12.011 2Harvard
The identification of mechanisms that regulate stem cell renewal has led to the development of innovative approaches to expand hematopoietic stem cells ex vivo. One approach reported by Wagner et al. (2016) in this issue of Cell Stem Cell has demonstrated dramatic expansion of umbilical cord blood stem cells that promote rapid engraftment while maintaining capacity for long-term hematopoiesis. Allogeneic hematopoietic stem cell transplantation (HSCT) is often the only curative therapy available for patients with high-risk leukemias or other hematologic cancers. HLA matching between recipient and donor is critical but only 20%– 30% of patients requiring allogeneic HSCT have HLA-matched sibling donors. Patients of Caucasian descent have a 70% chance of being paired with a fully HLA-matched, unrelated donor. However, this likelihood drops to 30% or less for patients with diverse ethnic backgrounds. This has led to the development of various approaches to identify alternate sources of allogeneic stem cells needed for the great majority of patients who can potentially benefit from allogeneic HSCT. Among these alternative sources, promising clinical results have been reported with partially HLA-mismatched unrelated donors, HLA-haplotype mismatched related donors, and partially HLA-matched umbilical cord blood (UCB) stem cells. In the 25 years since the first successful UCB transplant was reported, over 30,000 UCBTs have been performed with over 500,000 UCB units donated for public use in more than 100 international cord blood banks (Ballen et al., 2013). This extensive experience has demonstrated that cryopreserved UCB products contain sufficient numbers of hematopoietic stem cells (HSCs) for long-term hematopoietic reconstitution and successful UCB transplantation can be achieved despite some degree of HLA mismatching. Nevertheless, UCB units contain fewer HSCs than either bone marrow or mobilized peripheral blood stem cell products and this results in de-
layed hematopoietic recovery and immune reconstitution. Total nucleated cell (TNC) dose and the degree of HLA disparity between recipient and UCB unit correlate with rapidity of neutrophil and platelet recovery (Gluckman et al., 2004). Stem cell dose, quantified by CD34+ cell content or hematopoieticcolony-forming cells/units (CFUs), also correlates with rate of engraftment, nonrelapse mortality, and survival (Page et al., 2011). As a result, current recommendations are for a minimum TNC dose of 2.0–3.5 3 107/kg and >1.7 3 105 CD34+ cells/graft. To achieve the recommended minimum UCB cell dose, most adult patients receive two partially HLA-mismatched UCB units for transplantation. Several studies have suggested that double UCB transplants (dUCBTs) enhance early myeloid engraftment without an increased risk of graft versus host disease (GVHD) or non-relapse mortality. However, long-term hematopoiesis usually derives from only one of the two UCB units. Immune reconstitution remains delayed after dUCBT and these patients remain susceptible to opportunistic infections for prolonged periods. The use of two UCB units for transplantation has facilitated clinical evaluation of innovative approaches to enhance engraftment and persistence of UCB stem cells through ex vivo manipulation. In this setting, only one of the two UCB products is modified ex vivo and the second unit is infused without manipulation. Since UCB units are genetically distinct and partially HLA-mismatched to each other as well as to the recipient, it is relatively easy to monitor and compare
10 Cell Stem Cell 18, January 7, 2016 ª2016 Elsevier Inc.
engraftment, lineage differentiation, and long-term persistence of each stem cell product. In this issue of Cell Stem Cell, Wagner et al. (2016) report the results of a clinical trial where this approach is used to evaluate the ability of StemRegenin-1 (SR-1) to expand UCB stem cells in vitro (Wagner et al., 2016). SR-1 inhibits the aryl hydrocarbon receptor, thus blocking HSC differentiation. When combined with stem cell factor, FLT-3 ligand, thrombopoietin, and interleukin-6, this results in a remarkable median 330-fold expansion of CD34+ HSCs and median 854-fold increase in TNCs after 15 days of in vitro treatment. Seventeen patients received an untreated UCB product and, 4 hr later, an infusion of SR-1 expanded UCB stem cells from a second product. When compared with 111 dUCBT patients who received the same conditioning regimen and post-transplant immune suppression (historical control), engraftment of both granulocytes and platelets was significantly faster and hospital stay was significantly shorter in patients who received SR-1 expanded stem cells. Notably, this was accomplished without increased amounts of GVHD or transplant-associated toxicities. The only potentially adverse outcome in patients who were engrafted with SR-1-treated stem cells was delayed CD4 T cell recovery after transplant. When long-term engraftment was examined, the authors found that SR-1-treated stem cells were more likely to have long-term persistence than untreated stem cells, but long-term persistence of SR-1-treated stem cells was not observed in 6 of 17 patients. Further studies in some of these patients suggest
Cell Stem Cell
In Translation Table 1. Current Approaches to Ex Vivo Manipulation of UCB Stem Cells in Clinical Trials Median Days Ex Vivo to Myeloid Processing Engraftment
Long-term Engraftment by Immune Treated UCB Reconstitution Reference
Ex Vivo Treatment
Biologic Rationale
Ex Vivo Expansion
Tetraethylene pentamine (SCF, FLT-3L, TPO, IL-6)
copper chelation blocks HSC differentiation
TNC +++, CD34 +, CFUs ++
21 days
granulocytes: 30; not evaluated platelets: 48
not evaluated
(de Lima et al., 2008)
Notch ligand Delta 1 (fibronectin, SCT, FLT-3L, TPO, IL-6, IL-3)
provides HSC proliferative signal
TNC +++, CD34 +++
16 days
granulocytes: 13; <5% platelets: 38
not evaluated
(Delaney et al., 2010)
Mesenchymal MSCs provide signals TNC +, stromal cells (SCF, for HSC expansion CD34 ++, FLT3L, TPO, G-CSF) CFUs ++
14 days
granulocytes: 15; <10% platelets: 42
not evaluated
(de Lima et al., 2012)
16,16-dimethyl prostaglandin E2
facilitates HSC homing, proliferation, and self-renewal
N/A
2 hr
granulocytes: 18; 83% platelets: 43
delayed
(Cutler et al., 2013)
Nicotinamide (SCF, FLT-3L, TPO, IL-6)
inhibits HSC differentiation; facilitates HSC homing
TNC +++, CD34 ++
21 days
granulocytes: 13; 60%–80% platelets: 33
not evaluated
(Horwitz et al., 2014)
Fucosylation
facilitates HSC homing
N/A
30 min
granulocytes: 17; 50% platelets: 35
not evaluated
(Popat et al., 2015)
StemRegenin-1 (SCF, FLT-3L, TPO, IL-6)
SR-1 inhibition of aryl hydrocarbon receptor blocks HSC differentiation
TNC +++, 15 days CD34 +++, CFUs +++
granulocytes: 15; 65% platelets: 49
delayed
(Wagner et al., 2016)
SCT, stem cell factor; FLT-3L, Fms-related tyrosine kinase 3 ligand; TPO, thrombopoietin; IL-6, interleukin-6; G-CSF, granulocyte colony-stimulating factor; TNC, total nucleated cells; N/A, not applicable.
that this was due to immunologic rejection of SR-1-treated stem cells by T cells derived from the unmanipulated UCB unit. As noted by the authors, these findings have broad implications for UCB banking and our ability to identify suitable UCB products for patients without HLA-matched donors. Given the massive stem cell expansion achieved after SR-1 treatment, it may be possible to utilize cord blood collections currently felt to be inadequate. This would dramatically increase the number of UCB products available for transplantation as well as the likelihood of finding a more closely HLA-matched unit. It may also be possible to simply use single UCB products in adult recipients, thereby lowering the cost of obtaining adequate stem cell products while also reducing potential immunologic toxicities resulting from interactions between two HLA-mismatched immune systems after transplant. While this approach has many advantages, the requirement for in vitro expansion of UCB stem cells also imposes some practical challenges for its widespread use, as GMP facilities are
needed to safely process HSCs for each patient. All of these issues will likely be addressed in future studies that will be needed to validate the encouraging findings in this first clinical trial. While the results of the study reported by Wagner et al. are promising, these data must be placed into the context of other clinical trials involving ex vivo UCB manipulation. Thus far, clinical results of six other approaches have been published. A brief overview of these studies is summarized in Table 1. All of the published studies are relatively small, and more definitive comparisons must await larger multi-center trials. Nevertheless, it is possible to make general comparisons and identify issues that will need further examination. Using a wide variety of innovative reagents that utilize highly variable processing conditions (30 min to 21 days), almost all studies report rapid granulocyte recovery. In UCB products that are expanded for 14 to 21 days, rapid neutrophil recovery likely reflects transplantation of relatively large numbers of committed myeloid progenitor cells resulting from in vitro expansion with he-
matopoietic growth factors. With UCB products that are treated for very short periods (30 min to 2 hr), early neutrophil recovery likely reflects increased stem cell homing to bone marrow niches after infusion. Notably, some approaches that promote rapid neutrophil recovery do not result in sustained engraftment of the treated stem cell product (e.g., mesenchymal stem cell co-culture and Notchligand-mediated expansion). With these treatments, expansion of committed myeloid progenitors occurs without maintenance of the pool of undifferentiated HSCs required for long-term engraftment. With these approaches, expanded UCB stem cells promote rapid granulocyte recovery, but additional stem cell products are needed to support long-term hematopoiesis after transplant. In contrast, treatment with SR-1 or nicotinamide appears to expand committed myeloid progenitors while also preserving the pool of undifferentiated HSCs. Unfortunately, immune reconstitution has not been carefully evaluated in many of these studies. Markedly delayed T cell recovery is a common feature of UCB transplants,
Cell Stem Cell 18, January 7, 2016 ª2016 Elsevier Inc. 11
Cell Stem Cell
In Translation leading to frequent infectious complications. Improvements in T cell recovery are clearly needed in these patients and this will be an important objective for further optimizing strategies for ex vivo UCB treatment.
de Lima, M., McMannis, J., Gee, A., Komanduri, K., Couriel, D., Andersson, B.S., Hosing, C., Khouri, I., Jones, R., Champlin, R., et al. (2008). Bone Marrow Transplant. 41, 771–778.
Horwitz, M.E., Chao, N.J., Rizzieri, D.A., Long, G.D., Sullivan, K.M., Gasparetto, C., Chute, J.P., Morris, A., McDonald, C., Waters-Pick, B., et al. (2014). J. Clin. Invest. 124, 3121–3128.
de Lima, M., McNiece, I., Robinson, S.N., Munsell, M., Eapen, M., Horowitz, M., Alousi, A., Saliba, R., McMannis, J.D., Kaur, I., et al. (2012). N. Engl. J. Med. 367, 2305–2315.
Page, K.M., Zhang, L., Mendizabal, A., Wease, S., Carter, S., Gentry, T., Balber, A.E., and Kurtzberg, J. (2011). Biol. Blood Marrow Transplant. 17, 1362– 1374.
Delaney, C., Heimfeld, S., Brashem-Stein, C., Voorhies, H., Manger, R.L., and Bernstein, I.D. (2010). Nat. Med. 16, 232–236.
Popat, U., Mehta, R.S., Rezvani, K., Fox, P., Kondo, K., Marin, D., McNiece, I., Oran, B., Hosing, C., Olson, A., et al. (2015). Blood 125, 2885– 2892.
Gluckman, E., Rocha, V., Arcese, W., Michel, G., Sanz, G., Chan, K.W., Takahashi, T.A., Ortega, J., Filipovich, A., Locatelli, F., et al.; Eurocord Group (2004). Exp. Hematol. 32, 397–407.
Wagner, J.E., Jr., Brunstein, C.G., Boitano, A.E., DeFor, T.E., McKenna, D., Sumstad, D., Blazar, B.R., Tolar, J., Le, C., Jones, J., et al. (2016). Cell Stem Cell 18, this issue, 144–155.
REFERENCES Ballen, K.K., Gluckman, E., and Broxmeyer, H.E. (2013). Blood 122, 491–498. Cutler, C., Multani, P., Robbins, D., Kim, H.T., Le, T., Hoggatt, J., Pelus, L.M., Desponts, C., Chen, Y.B., Rezner, B., et al. (2013). Blood 122, 3074– 3081.
12 Cell Stem Cell 18, January 7, 2016 ª2016 Elsevier Inc.
In Translation Dramatic Expansion of HSCs: New Possibilities for HSC Transplants? Sarah Nikiforow1,2 and Jerome Ritz1,2,* 1Division
of Hematologic Malignancies, Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA Medical School, Boston, MA 02115, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.stem.2015.12.011 2Harvard
The identification of mechanisms that regulate stem cell renewal has led to the development of innovative approaches to expand hematopoietic stem cells ex vivo. One approach reported by Wagner et al. (2016) in this issue of Cell Stem Cell has demonstrated dramatic expansion of umbilical cord blood stem cells that promote rapid engraftment while maintaining capacity for long-term hematopoiesis. Allogeneic hematopoietic stem cell transplantation (HSCT) is often the only curative therapy available for patients with high-risk leukemias or other hematologic cancers. HLA matching between recipient and donor is critical but only 20%– 30% of patients requiring allogeneic HSCT have HLA-matched sibling donors. Patients of Caucasian descent have a 70% chance of being paired with a fully HLA-matched, unrelated donor. However, this likelihood drops to 30% or less for patients with diverse ethnic backgrounds. This has led to the development of various approaches to identify alternate sources of allogeneic stem cells needed for the great majority of patients who can potentially benefit from allogeneic HSCT. Among these alternative sources, promising clinical results have been reported with partially HLA-mismatched unrelated donors, HLA-haplotype mismatched related donors, and partially HLA-matched umbilical cord blood (UCB) stem cells. In the 25 years since the first successful UCB transplant was reported, over 30,000 UCBTs have been performed with over 500,000 UCB units donated for public use in more than 100 international cord blood banks (Ballen et al., 2013). This extensive experience has demonstrated that cryopreserved UCB products contain sufficient numbers of hematopoietic stem cells (HSCs) for long-term hematopoietic reconstitution and successful UCB transplantation can be achieved despite some degree of HLA mismatching. Nevertheless, UCB units contain fewer HSCs than either bone marrow or mobilized peripheral blood stem cell products and this results in de-
layed hematopoietic recovery and immune reconstitution. Total nucleated cell (TNC) dose and the degree of HLA disparity between recipient and UCB unit correlate with rapidity of neutrophil and platelet recovery (Gluckman et al., 2004). Stem cell dose, quantified by CD34+ cell content or hematopoieticcolony-forming cells/units (CFUs), also correlates with rate of engraftment, nonrelapse mortality, and survival (Page et al., 2011). As a result, current recommendations are for a minimum TNC dose of 2.0–3.5 3 107/kg and >1.7 3 105 CD34+ cells/graft. To achieve the recommended minimum UCB cell dose, most adult patients receive two partially HLA-mismatched UCB units for transplantation. Several studies have suggested that double UCB transplants (dUCBTs) enhance early myeloid engraftment without an increased risk of graft versus host disease (GVHD) or non-relapse mortality. However, long-term hematopoiesis usually derives from only one of the two UCB units. Immune reconstitution remains delayed after dUCBT and these patients remain susceptible to opportunistic infections for prolonged periods. The use of two UCB units for transplantation has facilitated clinical evaluation of innovative approaches to enhance engraftment and persistence of UCB stem cells through ex vivo manipulation. In this setting, only one of the two UCB products is modified ex vivo and the second unit is infused without manipulation. Since UCB units are genetically distinct and partially HLA-mismatched to each other as well as to the recipient, it is relatively easy to monitor and compare
10 Cell Stem Cell 18, January 7, 2016 ª2016 Elsevier Inc.
engraftment, lineage differentiation, and long-term persistence of each stem cell product. In this issue of Cell Stem Cell, Wagner et al. (2016) report the results of a clinical trial where this approach is used to evaluate the ability of StemRegenin-1 (SR-1) to expand UCB stem cells in vitro (Wagner et al., 2016). SR-1 inhibits the aryl hydrocarbon receptor, thus blocking HSC differentiation. When combined with stem cell factor, FLT-3 ligand, thrombopoietin, and interleukin-6, this results in a remarkable median 330-fold expansion of CD34+ HSCs and median 854-fold increase in TNCs after 15 days of in vitro treatment. Seventeen patients received an untreated UCB product and, 4 hr later, an infusion of SR-1 expanded UCB stem cells from a second product. When compared with 111 dUCBT patients who received the same conditioning regimen and post-transplant immune suppression (historical control), engraftment of both granulocytes and platelets was significantly faster and hospital stay was significantly shorter in patients who received SR-1 expanded stem cells. Notably, this was accomplished without increased amounts of GVHD or transplant-associated toxicities. The only potentially adverse outcome in patients who were engrafted with SR-1-treated stem cells was delayed CD4 T cell recovery after transplant. When long-term engraftment was examined, the authors found that SR-1-treated stem cells were more likely to have long-term persistence than untreated stem cells, but long-term persistence of SR-1-treated stem cells was not observed in 6 of 17 patients. Further studies in some of these patients suggest
Cell Stem Cell
In Translation Table 1. Current Approaches to Ex Vivo Manipulation of UCB Stem Cells in Clinical Trials Median Days Ex Vivo to Myeloid Processing Engraftment
Long-term Engraftment by Immune Treated UCB Reconstitution Reference
Ex Vivo Treatment
Biologic Rationale
Ex Vivo Expansion
Tetraethylene pentamine (SCF, FLT-3L, TPO, IL-6)
copper chelation blocks HSC differentiation
TNC +++, CD34 +, CFUs ++
21 days
granulocytes: 30; not evaluated platelets: 48
not evaluated
(de Lima et al., 2008)
Notch ligand Delta 1 (fibronectin, SCT, FLT-3L, TPO, IL-6, IL-3)
provides HSC proliferative signal
TNC +++, CD34 +++
16 days
granulocytes: 13; <5% platelets: 38
not evaluated
(Delaney et al., 2010)
Mesenchymal MSCs provide signals TNC +, stromal cells (SCF, for HSC expansion CD34 ++, FLT3L, TPO, G-CSF) CFUs ++
14 days
granulocytes: 15; <10% platelets: 42
not evaluated
(de Lima et al., 2012)
16,16-dimethyl prostaglandin E2
facilitates HSC homing, proliferation, and self-renewal
N/A
2 hr
granulocytes: 18; 83% platelets: 43
delayed
(Cutler et al., 2013)
Nicotinamide (SCF, FLT-3L, TPO, IL-6)
inhibits HSC differentiation; facilitates HSC homing
TNC +++, CD34 ++
21 days
granulocytes: 13; 60%–80% platelets: 33
not evaluated
(Horwitz et al., 2014)
Fucosylation
facilitates HSC homing
N/A
30 min
granulocytes: 17; 50% platelets: 35
not evaluated
(Popat et al., 2015)
StemRegenin-1 (SCF, FLT-3L, TPO, IL-6)
SR-1 inhibition of aryl hydrocarbon receptor blocks HSC differentiation
TNC +++, 15 days CD34 +++, CFUs +++
granulocytes: 15; 65% platelets: 49
delayed
(Wagner et al., 2016)
SCT, stem cell factor; FLT-3L, Fms-related tyrosine kinase 3 ligand; TPO, thrombopoietin; IL-6, interleukin-6; G-CSF, granulocyte colony-stimulating factor; TNC, total nucleated cells; N/A, not applicable.
that this was due to immunologic rejection of SR-1-treated stem cells by T cells derived from the unmanipulated UCB unit. As noted by the authors, these findings have broad implications for UCB banking and our ability to identify suitable UCB products for patients without HLA-matched donors. Given the massive stem cell expansion achieved after SR-1 treatment, it may be possible to utilize cord blood collections currently felt to be inadequate. This would dramatically increase the number of UCB products available for transplantation as well as the likelihood of finding a more closely HLA-matched unit. It may also be possible to simply use single UCB products in adult recipients, thereby lowering the cost of obtaining adequate stem cell products while also reducing potential immunologic toxicities resulting from interactions between two HLA-mismatched immune systems after transplant. While this approach has many advantages, the requirement for in vitro expansion of UCB stem cells also imposes some practical challenges for its widespread use, as GMP facilities are
needed to safely process HSCs for each patient. All of these issues will likely be addressed in future studies that will be needed to validate the encouraging findings in this first clinical trial. While the results of the study reported by Wagner et al. are promising, these data must be placed into the context of other clinical trials involving ex vivo UCB manipulation. Thus far, clinical results of six other approaches have been published. A brief overview of these studies is summarized in Table 1. All of the published studies are relatively small, and more definitive comparisons must await larger multi-center trials. Nevertheless, it is possible to make general comparisons and identify issues that will need further examination. Using a wide variety of innovative reagents that utilize highly variable processing conditions (30 min to 21 days), almost all studies report rapid granulocyte recovery. In UCB products that are expanded for 14 to 21 days, rapid neutrophil recovery likely reflects transplantation of relatively large numbers of committed myeloid progenitor cells resulting from in vitro expansion with he-
matopoietic growth factors. With UCB products that are treated for very short periods (30 min to 2 hr), early neutrophil recovery likely reflects increased stem cell homing to bone marrow niches after infusion. Notably, some approaches that promote rapid neutrophil recovery do not result in sustained engraftment of the treated stem cell product (e.g., mesenchymal stem cell co-culture and Notchligand-mediated expansion). With these treatments, expansion of committed myeloid progenitors occurs without maintenance of the pool of undifferentiated HSCs required for long-term engraftment. With these approaches, expanded UCB stem cells promote rapid granulocyte recovery, but additional stem cell products are needed to support long-term hematopoiesis after transplant. In contrast, treatment with SR-1 or nicotinamide appears to expand committed myeloid progenitors while also preserving the pool of undifferentiated HSCs. Unfortunately, immune reconstitution has not been carefully evaluated in many of these studies. Markedly delayed T cell recovery is a common feature of UCB transplants,
Cell Stem Cell 18, January 7, 2016 ª2016 Elsevier Inc. 11
Cell Stem Cell
In Translation leading to frequent infectious complications. Improvements in T cell recovery are clearly needed in these patients and this will be an important objective for further optimizing strategies for ex vivo UCB treatment.
de Lima, M., McMannis, J., Gee, A., Komanduri, K., Couriel, D., Andersson, B.S., Hosing, C., Khouri, I., Jones, R., Champlin, R., et al. (2008). Bone Marrow Transplant. 41, 771–778.
Horwitz, M.E., Chao, N.J., Rizzieri, D.A., Long, G.D., Sullivan, K.M., Gasparetto, C., Chute, J.P., Morris, A., McDonald, C., Waters-Pick, B., et al. (2014). J. Clin. Invest. 124, 3121–3128.
de Lima, M., McNiece, I., Robinson, S.N., Munsell, M., Eapen, M., Horowitz, M., Alousi, A., Saliba, R., McMannis, J.D., Kaur, I., et al. (2012). N. Engl. J. Med. 367, 2305–2315.
Page, K.M., Zhang, L., Mendizabal, A., Wease, S., Carter, S., Gentry, T., Balber, A.E., and Kurtzberg, J. (2011). Biol. Blood Marrow Transplant. 17, 1362– 1374.
Delaney, C., Heimfeld, S., Brashem-Stein, C., Voorhies, H., Manger, R.L., and Bernstein, I.D. (2010). Nat. Med. 16, 232–236.
Popat, U., Mehta, R.S., Rezvani, K., Fox, P., Kondo, K., Marin, D., McNiece, I., Oran, B., Hosing, C., Olson, A., et al. (2015). Blood 125, 2885– 2892.
Gluckman, E., Rocha, V., Arcese, W., Michel, G., Sanz, G., Chan, K.W., Takahashi, T.A., Ortega, J., Filipovich, A., Locatelli, F., et al.; Eurocord Group (2004). Exp. Hematol. 32, 397–407.
Wagner, J.E., Jr., Brunstein, C.G., Boitano, A.E., DeFor, T.E., McKenna, D., Sumstad, D., Blazar, B.R., Tolar, J., Le, C., Jones, J., et al. (2016). Cell Stem Cell 18, this issue, 144–155.
REFERENCES Ballen, K.K., Gluckman, E., and Broxmeyer, H.E. (2013). Blood 122, 491–498. Cutler, C., Multani, P., Robbins, D., Kim, H.T., Le, T., Hoggatt, J., Pelus, L.M., Desponts, C., Chen, Y.B., Rezner, B., et al. (2013). Blood 122, 3074– 3081.
12 Cell Stem Cell 18, January 7, 2016 ª2016 Elsevier Inc.