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

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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.

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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.

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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

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.

Correspondence to Shaoyi Jiang or Colleen Delaney.

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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.

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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.

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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

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