Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Expansion of primitive human hematopoietic stem cells by culture in a zwitterionic hydrogel

Abstract

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: A biodegradable zwitterionic hydrogel promotes the expansion of CD34+ CB progenitor cells.
Fig. 2: Culture in zwitterionic hydrogel inhibits the differentiation of CD34+ CB progenitors.
Fig. 3: The ZTGopt culture condition promotes expansion of LT-HSCs from CD34+ CB progenitors.
Fig. 4: 3D zwitterionic culture avoids excessive ROS production and nonspecific-signaling-pathway activity.
Fig. 5: 3D zwitterionic culture results in reduced metabolic activity in CD34+ CB progenitors.
Fig. 6: RNA expression analysis indicates that HSPCs cultured in the ZTGopt culture condition are not activated for differentiation.

Data availability

The data that support the findings of this study are available within the paper and its Supplementary Information files. The RNA-seq data that support the findings of this study are publicly accessible from the National Center for Biotechnology Information database with the accession code GSE85800.

References

  1. Long, Y. C. & Zierath, J. R. AMP-activated protein kinase signaling in metabolic regulation. J. Clin. Invest. 116, 1776–1783 (2006).

    Article  CAS  Google Scholar 

  2. Gragert, L. et al. HLA match likelihoods for hematopoietic stem-cell grafts in the US registry. N. Engl. J. Med. 371, 339–348 (2014).

    Article  CAS  Google Scholar 

  3. Milano, F. et al. Cord-blood transplantation in patients with minimal residual disease. N. Engl. J. Med. 375, 944–953 (2016).

    Article  Google Scholar 

  4. Ballen, K. K., Gluckman, E. & Broxmeyer, H. E. Umbilical cord blood transplantation: the first 25 years and beyond. Blood 122, 491–498 (2013).

    Article  CAS  Google Scholar 

  5. Dahlberg, A., Delaney, C. & Bernstein, I. D. Ex vivo expansion of human hematopoietic stem and progenitor cells. Blood 117, 6083–6090 (2011).

    Article  CAS  Google Scholar 

  6. 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).

    Article  CAS  Google Scholar 

  7. Ratajczak, M. Z. Phenotypic and functional characterization of hematopoietic stem cells. Curr. Opin. Hematol. 15, 293–300 (2008).

    Article  Google Scholar 

  8. Pineault, N. & Abu-Khader, A. Advances in umbilical cord blood stem cell expansion and clinical translation. Exp. Hematol. 43, 498–513 (2015).

    Article  Google Scholar 

  9. 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).

    Article  CAS  Google Scholar 

  10. Delaney, C. et al. Notch-mediated expansion of human cord blood progenitor cells capable of rapid myeloid reconstitution. Nat. Med. 16, 232–236 (2010).

    Article  CAS  Google Scholar 

  11. 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).

    Article  Google Scholar 

  12. Boitano, A. E. et al. Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells. Science 329, 1345–1348 (2010).

    Article  CAS  Google Scholar 

  13. Fares, I. et al. Pyrimidoindole derivatives are agonists of human hematopoietic stem cell self-renewal. Science 345, 1509–1512 (2014).

    Article  CAS  Google Scholar 

  14. Bretscher, M. S. Mammalian plasma membranes. Nature 258, 43–49 (1975).

    Article  CAS  Google Scholar 

  15. 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).

    Article  CAS  Google Scholar 

  16. Liu, W. F. et al. Real-time in vivo detection of biomaterial-induced reactive oxygen species. Biomaterials 32, 1796–1801 (2011).

    Article  Google Scholar 

  17. 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).

    Article  CAS  Google Scholar 

  18. Bigarella, C. L., Liang, R. & Ghaffari, S. Stem cells and the impact of ROS signaling. Development 141, 4206–4218 (2014).

    Article  CAS  Google Scholar 

  19. Jiang, S. & Cao, Z. Ultralow‐fouling, functionalizable, and hydrolyzable zwitterionic materials and their derivatives for biological applications. Adv. Mater. 22, 920–932 (2010).

    Article  CAS  Google Scholar 

  20. Keefe, A. J. & Jiang, S. Poly (zwitterionic) protein conjugates offer increased stability without sacrificing binding affinity or bioactivity. Nat. Chem. 4, 59 (2012).

    Article  CAS  Google Scholar 

  21. Zhang, L. et al. Zwitterionic hydrogels implanted in mice resist the foreign-body reaction. Nat. Biotech. 31, 553–556 (2013).

    Article  CAS  Google Scholar 

  22. Zhang, P. et al. Zwitterionic gel encapsulation promotes protein stability, enhances pharmacokinetics, and reduces immunogenicity. Proc. Natl. Acad. Sci. USA 112, 12046–12051 (2015).

    Article  CAS  Google Scholar 

  23. 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).

    Article  CAS  Google Scholar 

  24. 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).

    Article  CAS  Google Scholar 

  25. West, J. L. & Hubbell, J. A. Polymeric biomaterials with degradation sites for proteases involved in cell migration. Macromolecules 32, 241–244 (1999).

    Article  CAS  Google Scholar 

  26. 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).

    Article  CAS  Google Scholar 

  27. 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).

    Article  CAS  Google Scholar 

  28. Nel, A., Xia, T., Mädler, L. & Li, N. Toxic potential of materials at the nanolevel. Science 311, 622–627 (2006).

    Article  CAS  Google Scholar 

  29. 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).

    Article  CAS  Google Scholar 

  30. 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).

    Article  CAS  Google Scholar 

  31. Shin, S. Y. et al. Hydrogen peroxide negatively modulates Wnt signaling through downregulation of β-catenin. Cancer Lett. 212, 225–231 (2004).

    Article  CAS  Google Scholar 

  32. Reya, T. et al. A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature 423, 409–414 (2003).

    Article  CAS  Google Scholar 

  33. 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).

    Article  CAS  Google Scholar 

  34. 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).

    Article  CAS  Google Scholar 

  35. Ito, K. et al. Regulation of oxidative stress by ATM is required for self-renewal of haematopoietic stem cells. Nature 431, 997–1002 (2004).

    Article  CAS  Google Scholar 

  36. 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).

    Article  CAS  Google Scholar 

  37. 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).

    Article  CAS  Google Scholar 

  38. 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).

    Article  CAS  Google Scholar 

  39. 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).

    Article  CAS  Google Scholar 

  40. 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).

    Article  CAS  Google Scholar 

  41. 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.

    Article  Google Scholar 

  42. McGraw, T. E. & Mittal, V. Stem cells: Metabolism regulates differentiation. Nat. Chem. Biol. 6, 176–177 (2010).

    Article  CAS  Google Scholar 

  43. Ito, K. & Suda, T. Metabolic requirements for the maintenance of self-renewing stem cells. Nat. Rev. Mol. Cell Biol. 15, 243–256 (2014).

    Article  CAS  Google Scholar 

  44. 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).

    Article  CAS  Google Scholar 

  45. 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).

    Article  CAS  Google Scholar 

  46. 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).

    Article  CAS  Google Scholar 

  47. 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).

    Article  CAS  Google Scholar 

  48. 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).

    Article  Google Scholar 

  49. Antonchuk, J., Sauvageau, G. & Humphries, R. K. HOXB4-induced expansion of adult hematopoietic stem cells ex vivo. Cell 109, 39–45 (2002).

    Article  CAS  Google Scholar 

  50. 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).

    Article  CAS  Google Scholar 

  51. 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).

    Article  CAS  Google Scholar 

  52. 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).

    Article  CAS  Google Scholar 

  53. 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).

    Google Scholar 

  54. Trapnell, C., Pachter, L. & Salzberg, S. L. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009).

    Article  CAS  Google Scholar 

  55. Anders, S., Pyl, P. T. & Huber, W. HTSeq–a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2014).

    Article  Google Scholar 

  56. Rau, A., Gallopin, M., Celeux, G. & Jaffrézic, F. Data-based filtering for replicated high-throughput transcriptome sequencing experiments. Bioinformatics 29, 2146–2152 (2013).

    Article  CAS  Google Scholar 

  57. 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).

    Article  CAS  Google Scholar 

  58. Reiner, A., Yekutieli, D. & Benjamini, Y. Identifying differentially expressed genes using false discovery rate controlling procedures. Bioinformatics 19, 368–375 (2003).

    Article  CAS  Google Scholar 

  59. 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).

    Article  Google Scholar 

  60. Supek, F., Bošnjak, M., Škunca, N. & Šmuc, T. REVIGO summarizes and visualizes long lists of gene ontology terms. PloS One 6, e21800 (2011).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Contributions

C.D. and S.J. designed and supervised the studies. T.B., J.L., A.S., F.M., S.I., F.S., C.N., P.J., J.J.D., R.S.B., H.-C.H., B.L., M.B.O. and P. Z. conducted the experiments and data analysis. T.B., J.L., A.S., S.H., C.D. and S.J. wrote the paper. All authors read the paper and contributed to its final form.

Corresponding authors

Correspondence to Shaoyi Jiang or Colleen Delaney.

Ethics declarations

Competing interests

S.J. is a cofounder of Taproot Medical Technologies, LLC. C.D. is the founder and chief scientific officer of Nohla Therapeutics, Inc.

Additional information

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.

Extended data

Extended Data Fig. 1 Synthesis of zwitterionic and PEG star polymers.

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. df, 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. gi, 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.

Extended Data Fig. 3 Dynamic cell-cycle analysis.

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).

Extended Data Fig. 4 Effect of additional passages on expanded cells from ZTG culture.

a, Total cell number in ZTG culture, to day 44. bd, 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.

Extended Data Fig. 7 In vivo function of expanded BM-CD34+ HSPCs from ZTGopt culture.

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.

Extended Data Fig. 8 The effect of ROS scavenger NAC on the expansion behavior of HSPCs.

Freshly isolated HSPCs were seeded in ZTG, DXI and control conditions, and their culture medium was supplemented with different concentrations of NAC. ae, 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). fh, 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.

Extended Data Fig. 9 ZTG culture cannot reverse the impact of ROS on HSPCs.

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.

Extended Data Fig. 10 Dynamic metabolism measurement of HSPCs in different culture conditions.

ac, 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.

Supplementary information

Supplementary Information

Supplementary Figures 1–10

Reporting Summary

Supplementary Tables

Supplementary Tables 1–7

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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). https://doi.org/10.1038/s41591-019-0601-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41591-019-0601-5

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing