The ability to expand hematopoietic stem and progenitor cells (HSPCs) ex vivo is critical to fully realize the potential of HSPC-based therapies. In particular, the application of clinically effective therapies, such as cord blood transplantation, has been impeded because of limited HSPC availability. Here, using 3D culture of human HSPCs in a degradable zwitterionic hydrogel, we achieved substantial expansion of phenotypically primitive CD34+ cord blood and bone-marrow-derived HSPCs. This culture system led to a 73-fold increase in long-term hematopoietic stem cell (LT-HSC) frequency, as demonstrated by limiting dilution assays, and the expanded HSPCs were capable of hematopoietic reconstitution for at least 24 weeks in immunocompromised mice. Both the zwitterionic characteristics of the hydrogel and the 3D format were important for HSPC self-renewal. Mechanistically, the impact of 3D zwitterionic hydrogel culture on mitigating HSPC differentiation and promoting self-renewal might result from an inhibition of excessive reactive oxygen species (ROS) production via suppression of O2-related metabolism. HSPC expansion using zwitterionic hydrogels has the potential to facilitate the clinical application of hematopoietic-stem-cell therapies.
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Long, Y. C. & Zierath, J. R. AMP-activated protein kinase signaling in metabolic regulation. J. Clin. Invest. 116, 1776–1783 (2006).
Gragert, L. et al. HLA match likelihoods for hematopoietic stem-cell grafts in the US registry. N. Engl. J. Med. 371, 339–348 (2014).
Milano, F. et al. Cord-blood transplantation in patients with minimal residual disease. N. Engl. J. Med. 375, 944–953 (2016).
Ballen, K. K., Gluckman, E. & Broxmeyer, H. E. Umbilical cord blood transplantation: the first 25 years and beyond. Blood 122, 491–498 (2013).
Dahlberg, A., Delaney, C. & Bernstein, I. D. Ex vivo expansion of human hematopoietic stem and progenitor cells. Blood 117, 6083–6090 (2011).
Murray, L. J. et al. Thrombopoietin, flt3, and kit ligands together suppress apoptosis of human mobilized CD34+ cells and recruit primitive CD34+ Thy-1+ cells into rapid division. Exp. Hematol. 27, 1019–1028 (1999).
Ratajczak, M. Z. Phenotypic and functional characterization of hematopoietic stem cells. Curr. Opin. Hematol. 15, 293–300 (2008).
Pineault, N. & Abu-Khader, A. Advances in umbilical cord blood stem cell expansion and clinical translation. Exp. Hematol. 43, 498–513 (2015).
Csaszar, E. et al. Rapid expansion of human hematopoietic stem cells by automated control of inhibitory feedback signaling. Cell Stem Cell 10, 218–229 (2012).
Delaney, C. et al. Notch-mediated expansion of human cord blood progenitor cells capable of rapid myeloid reconstitution. Nat. Med. 16, 232–236 (2010).
Delaney, C. et al. Infusion of a non-HLA-matched ex-vivo expanded cord blood progenitor cell product after intensive acute myeloid leukaemia chemotherapy: a phase 1 trial. Lancet Hematol. 3, e330–9 (2016).
Boitano, A. E. et al. Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells. Science 329, 1345–1348 (2010).
Fares, I. et al. Pyrimidoindole derivatives are agonists of human hematopoietic stem cell self-renewal. Science 345, 1509–1512 (2014).
Bretscher, M. S. Mammalian plasma membranes. Nature 258, 43–49 (1975).
Cuchiara, M. L. et al. Bioactive poly (ethylene glycol) hydrogels to recapitulate the HSC niche and facilitate HSC expansion in culture. Biotechnol. Bioeng. 113, 870–881 (2016).
Liu, W. F. et al. Real-time in vivo detection of biomaterial-induced reactive oxygen species. Biomaterials 32, 1796–1801 (2011).
Yu, L., Shi, Z., Gao, L. & Li, C. Mitigated reactive oxygen species generation leads to an improvement of cell proliferation on poly [glycidyl methacrylate‐co‐poly (ethylene glycol) methacrylate] functionalized polydimethylsiloxane surfaces. J. Biomed. Mater. Res. A 103, 2987–2997 (2015).
Bigarella, C. L., Liang, R. & Ghaffari, S. Stem cells and the impact of ROS signaling. Development 141, 4206–4218 (2014).
Jiang, S. & Cao, Z. Ultralow‐fouling, functionalizable, and hydrolyzable zwitterionic materials and their derivatives for biological applications. Adv. Mater. 22, 920–932 (2010).
Keefe, A. J. & Jiang, S. Poly (zwitterionic) protein conjugates offer increased stability without sacrificing binding affinity or bioactivity. Nat. Chem. 4, 59 (2012).
Zhang, L. et al. Zwitterionic hydrogels implanted in mice resist the foreign-body reaction. Nat. Biotech. 31, 553–556 (2013).
Zhang, P. et al. Zwitterionic gel encapsulation promotes protein stability, enhances pharmacokinetics, and reduces immunogenicity. Proc. Natl. Acad. Sci. USA 112, 12046–12051 (2015).
DeForest, C. A., Polizzotti, B. D. & Anseth, K. S. Sequential click reactions for synthesizing and patterning three-dimensional cell microenvironments. Nat. Mater. 8, 659–664 (2009).
Anderson, S. B., Lin, C.-C., Kuntzler, D. V. & Anseth, K. S. The performance of human mesenchymal stem cells encapsulated in cell-degradable polymer-peptide hydrogels. Biomaterials 32, 3564–3574 (2011).
West, J. L. & Hubbell, J. A. Polymeric biomaterials with degradation sites for proteases involved in cell migration. Macromolecules 32, 241–244 (1999).
DeForest, C. A. & Anseth, K. S. Cytocompatible click-based hydrogels with dynamically tunable properties through orthogonal photoconjugation and photocleavage reactions. Nat. Chem. 3, 925–931 (2011).
Ito, M. et al. NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse model for engraftment of human cells. Blood 100, 3175–3182 (2002).
Nel, A., Xia, T., Mädler, L. & Li, N. Toxic potential of materials at the nanolevel. Science 311, 622–627 (2006).
Kaplan, S., Basford, R., Mora, E., Jeong, M. & Simmons, R. Biomaterial‐induced alterations of neutrophil superoxide production. J. Biomed. Mater. Res. 26, 1039–1051 (1992).
Ray, P. D., Huang, B.-W. & Tsuji, Y. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cell. Signal. 24, 981–990 (2012).
Shin, S. Y. et al. Hydrogen peroxide negatively modulates Wnt signaling through downregulation of β-catenin. Cancer Lett. 212, 225–231 (2004).
Reya, T. et al. A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature 423, 409–414 (2003).
Ito, K. et al. Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells. Nat. Med. 12, 446–451 (2006).
Yoshida, S. et al. Redox regulates mammalian target of rapamycin complex 1 (mTORC1) activity by modulating the TSC1/TSC2-Rheb GTPase pathway. J. Biol. Chem. 286, 32651–32660 (2011).
Ito, K. et al. Regulation of oxidative stress by ATM is required for self-renewal of haematopoietic stem cells. Nature 431, 997–1002 (2004).
Jang, Y.-Y. & Sharkis, S. J. A low level of reactive oxygen species selects for primitive hematopoietic stem cells that may reside in the low-oxygenic niche. Blood 110, 3056–3063 (2007).
Wang, Y., Kellner, J., Liu, L. & Zhou, D. Inhibition of p38 mitogen-activated protein kinase promotes ex vivo hematopoietic stem cell expansion. Stem Cells Dev. 20, 1143–1152 (2011).
Huang, J., Nguyen-McCarty, M., Hexner, E. O., Danet-Desnoyers, G. & Klein, P. S. Maintenance of hematopoietic stem cells through regulation of Wnt and mTOR pathways. Nat. Med. 18, 1778–1785 (2012).
Luo, Y. et al. Rapamycin enhances long-term hematopoietic reconstitution of ex vivo expanded mouse hematopoietic stem cells by inhibiting senescence. Transplantation 97, 20–9 (2014).
Fleming, H. E. et al. Wnt signaling in the niche enforces hematopoietic stem cell quiescence and is necessary to preserve self-renewal in vivo. Cell Stem Cell 2, 274–283 (2008).
de Almeida, M. J., Luchsinger, L. L., Corrigan, D. J., Williams, L. J. & Snoeck, H.-W. Dye-independent methods reveal elevated mitochondrial mass in hematopoietic stem cells. Cell Stem Cell 21, 725–729 (2017). e724.
McGraw, T. E. & Mittal, V. Stem cells: Metabolism regulates differentiation. Nat. Chem. Biol. 6, 176–177 (2010).
Ito, K. & Suda, T. Metabolic requirements for the maintenance of self-renewing stem cells. Nat. Rev. Mol. Cell Biol. 15, 243–256 (2014).
Folmes, C. D., Dzeja, P. P., Nelson, T. J. & Terzic, A. Metabolic plasticity in stem cell homeostasis and differentiation. Cell Stem Cell 11, 596–606 (2012).
Enver, T., Pera, M., Peterson, C. & Andrews, P. W. Stem cell states, fates, and the rules of attraction. Cell Stem Cell 4, 387–397 (2009).
Nishida, M. et al. Gα12/13-and reactive oxygen species-dependent activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase by angiotensin receptor stimulation in rat neonatal cardiomyocytes. J. Biol. Chem. 280, 18434–18441 (2005).
Carey, A. L. et al. Interleukin-6 increases insulin-stimulated glucose disposal in humans and glucose uptake and fatty acid oxidation in vitro via AMP-activated protein kinase. Diabetes 55, 2688–2697 (2006).
De Lima, M. et al. Transplantation of ex vivo expanded cord blood cells using the copper chelator tetraethylenepentamine: a phase I/II clinical trial. Bone Marrow Transplant. 41, 771–778 (2008).
Antonchuk, J., Sauvageau, G. & Humphries, R. K. HOXB4-induced expansion of adult hematopoietic stem cells ex vivo. Cell 109, 39–45 (2002).
Lutz, J.-F., Börner, H. G. & Weichenhan, K. Combining ATRP and “click” chemistry: a promising platform toward functional biocompatible polymers and polymer bioconjugates. Macromolecules 39, 6376–6383 (2006).
Zhang, Z., Chen, S. F., Chang, Y. & Jiang, S. Y. Surface grafted sulfobetaine polymers via atom transfer radical polymerization as superlow fouling coatings. J. Phys. Chem. B 110, 10799–10804 (2006).
Shen, Y.-I. et al. Hyaluronic acid hydrogel stiffness and oxygen tension affect cancer cell fate and endothelial sprouting. Biomater. Sci. 2, 655–665 (2014).
Hu, Y. & Smyth, G. K. ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J. Immunol. Methods 347, (70–78 (2009).
Trapnell, C., Pachter, L. & Salzberg, S. L. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009).
Anders, S., Pyl, P. T. & Huber, W. HTSeq–a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2014).
Rau, A., Gallopin, M., Celeux, G. & Jaffrézic, F. Data-based filtering for replicated high-throughput transcriptome sequencing experiments. Bioinformatics 29, 2146–2152 (2013).
Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).
Reiner, A., Yekutieli, D. & Benjamini, Y. Identifying differentially expressed genes using false discovery rate controlling procedures. Bioinformatics 19, 368–375 (2003).
Young, M. D., Wakefield, M. J., Smyth, G. K. & Oshlack, A. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol. 11, 1 (2010).
Supek, F., Bošnjak, M., Škunca, N. & Šmuc, T. REVIGO summarizes and visualizes long lists of gene ontology terms. PloS One 6, e21800 (2011).
T.B., A.S., P.Z. and S.J. were supported by National Science Foundation (DMR 1307375). F.S., B.L. and S.J. were supported by National Science Foundation (CBET-1264477). P.J., H.-C.H. and S.J. were supported by the Office of Naval Research (N00014-14-1-0090 and N00014-15-1-2277). M.B.O. and S.J. were supported by the University of Washington. S.H. was supported by NIDDK grant (DK 106829). J.J.D. and R.S.B. were supported by NIH/NCI Cancer Center Support Grant (P30 CA015704). We thank D. Raftery and H. Gu from the Northwest Metabolomics Research Center (NW-MRC) for assisting with LC–MS assessment. We also thank all the mothers who donated their cord blood for research, without whom this work would not have been possible.
S.J. is a cofounder of Taproot Medical Technologies, LLC. C.D. is the founder and chief scientific officer of Nohla Therapeutics, Inc.
Peer review information Michael Basson was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
A, N3-terminated star-shaped pCBAA was produced by atom-transfer radical-polymerization and subsequent azide substitution. Next, the terminal azide groups were converted to NH2 groups using a ‘click’ reaction. Finally, DIFO3 was functionalized to the end of pCBAA star polymers via EDC–NHS reaction. The scheme shows reactions on one representative arm of the four-arm star-shaped pCBAA synthesized, with the other equivalent arms denoted as X1, X2, and so on, at each step. B, Synthesis of functionalized PEG star polymers. The DIFO3 group was functionalized to the end of PEG star polymers via EDCNHS chemistry.
Extended Data Fig. 2 Optimization of 3D ZTG culture for ex vivo HSPC expansion, and the effect of growth factors, seeding density and hydrogel stiffness on the expansion, viability and CD34 purity of expanded cells in ZTG culture.
a–c, ZTG-encapsulated CB CD34+ HSPCs were cultured in SFEM II medium supplemented with three growth factors (SCF, FLT3 and TPO), five growth factors (SCF, FLT3, TPO, IL-3 and IL-6) or no growth factors. The seeding density was fixed at 1.2 × 107 cells per ml, and the stiffness of the hydrogel was fixed at 0.7 kPa. We dynamically monitored the fold expansion (a), viability (b) and CD34+ population purity (c) in each condition. The five growth factors condition (5-GF) was selected as the optimal cytokine condition. d–f, ZTG-encapsulated cord blood CD34+ HSPCs were cultured at seeding densities of 0.6 × 107 cells per ml, 1.2 × 107 cells per ml or 2.5 × 107 cells per ml. SFEM II medium supplemented with five growth factors (SCF, FLT3, TPO, IL-3 and IL-6) was used and the stiffness of the hydrogel fixed at 0.7 kPa. We dynamically monitored the fold expansion (d), viability (e) and CD34+ population purity (f) in each condition. 1.2 × 107 cells ml−1 was selected as the optimal seeding density. g–i, CB CD34+ HSPCs were encapsulated and cultured in ZTG hydrogels with different stiffness (ZTGopt (0.7 kPa), ZTGmedium (1.9 kPa), and ZTGhigh (5 kPa); softer hydrogels presented handling difficulties). SFEM II medium that was supplemented with five growth factors (SCF, FLT3, TPO, IL-3 and IL-6) was used, and the seeding density was fixed at 1.2 × 107 cells per ml. We dynamically monitored the fold expansion (g), viability (h) and CD34+ population purity (i) in each condition, and 0.7 kPa was selected as the optimal hydrogel stiffness. ns indicates no significant difference. Values represent mean ± s.d., n = 3 biologically independent experiments and the two-tailed Student’s t-test P values are indicated.
a, Representative phase-contrast images of ZTGopt cells at different time points. Scale bar, 250 µm. b, Dynamic cell-cycle analysis by FACS using anti-Ki-67 and Hoechst 33342 staining for HSPCs in ZTGopt culture. c, Dynamic change of cell-cycle subsets after transferring cells before (Fresh) and after ZTGopt culture into control conditions. ZTGopt cells show delayed entry into the cell cycle. Mean ± s.d. is shown; n = 3 biologically independent experiments. **P < 0.001, ***P < 0.0005 (two-tailed Student’s t test).
a, Total cell number in ZTG culture, to day 44. b–d, Mean fluorescence intensity of CD34 (b), CD45RA (c) and lineage marker cocktail (d) at different time points. Values represent means ± s.d., n = 3 biologically independent experiments and the two-tailed Student’s t-test P values are indicated.
Extended Data Fig. 5 Significant differentiation in PEG hydrogels leads to lower fold expansion of CD34+ cells after 24 d.
a, The modulus of ZTG and PEG hydrogels at day 0. b, The modulus loss of ZTG and PEG hydrogels at day 14. c, Percentage of CD34+ cells after culture in ZTG and PEG hydrogels for 14 and 24 d. d, Fold expansion of CD34+ cells after culture in ZTG and PEG hydrogels for 14 and 24 d. e, Dissolved oxygen (DO) levels measured after 24-h culture in each system. f, MFI of cellular ROS level in each culture at culture day 1. Values represent mean ± s.d., n = 3 biologically independent experiments and the two-tailed Student’s t-test P values are indicated.
Extended Data Fig. 6 Zwitterionic hydrogel restrains the differentiation of bone marrow HSPCs during ex vivo expansion.
a,b, Representative flow cytometry dot plots for fresh (a) and ZTGopt cultured (b) populations. Experiments were repeated three times. FSC, forward scatter; SSC, side scatter. c, Fold expansion of total and CD34+ cells after ZTGopt culture. d, CFU numbers per 1,000 fresh cells or total ZTGopt cultured progeny of 1,000 starting cells. Values represent mean ± s.d., n = 3 independent experiments and the two-tailed Student’s t-test P values are indicated.
Levels of human engraftment and lineage repopulation in NSG mice transplanted with different actual cell doses, at week 24 post-transplant. The frequencies of human engraftment (CD45+) in NSG BM samples were examined by flow cytometry. Among these CD45+ human cells, the frequencies of human lineage cells (CD45+CD33+, CD45+CD19+, CD45+CD3+, CD45+CD56+ and CD45+CD34+) were examined. Mouse cells were not considered when calculating the frequencies of human lineage cells. The cell doses on the x axes indicate the actual number of injected cells. Horizontal lines indicate the average value for each group. n = 3 biologically independent experiments were performed with similar results, and representative plots were shown.
Freshly isolated HSPCs were seeded in ZTG, DXI and control conditions, and their culture medium was supplemented with different concentrations of NAC. a–e, We analyzed cellular ROS level (a) and ROS-related signaling pathway p16 (b), p38 (c), mTOR (d), and Wnt–β-catenin (e) after 1-d culture. We found a significant difference between ZTG and the two control cultures in all conditions (P < 0.001, two-tailed Student’s t test). f–h, Cells were then cultured in each condition for 14 d, and the primitive HSPC population (CD34+CD45RA−) purity (f), cell viability (g) and fold change (h) were analyzed. Values represent mean ± s.d., n = 3 biologically independent experiments.
Freshly isolated HSPCs were treated with H2O2 for 6 hours. Prior to ZTG encapsulation, cells were analyzed for apoptosis (a), intracellular ROS level (b) and MFIs of phospho-p38, phospho-mTOR and β-catenin (c) (n = 3 biologically independent experiments were repeated with similar results). d, Cells were then cultured in ZTG gels for 14 d, and the primitive HSPC population (CD34+CD45RA–) was analyzed. Values represent mean ± s.d., n = 3 biologically independent experiments and the two-tailed Student’s t-test P value is indicated.
a–c, Dynamic change in glucose consumption (a), lactate secretion (b) and amino acid metabolism (c) of CB cells after ex vivo culture in each condition. Values represent mean ± s.d., n = 3 biologically independent experiments are indicated.
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Bai, T., Li, J., Sinclair, A. et al. Expansion of primitive human hematopoietic stem cells by culture in a zwitterionic hydrogel. Nat Med 25, 1566–1575 (2019) doi:10.1038/s41591-019-0601-5