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Effective Labeling of Primary Somatic Stem Cells with BaTiO3 Nanocrystals for Second Harmonic Generation Imaging

Nami Sugiyama, Ali Y. Sonay, Roxanne Tussiwand, Bruce E. Cohen, and Periklis Pantazis*

Dr. N. Sugiyama, A. Y. Sonay, Prof. P. PantazisDepartment of Biosystems Science and Engineering (D-BSSE)Eidgenössische Technische Hochschule (ETH) Zurich4058 Basel, SwitzerlandE-mail: periklis.pantazis@bsse.ethz.chProf. R. TussiwandDepartment of BiomedicineUniversity of Basel4058 Basel, SwitzerlandDr. B. E. CohenThe Molecular FoundryLawrence Berkeley National LaboratoryBerkeley, CA 94720, USA

The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/smll.201703386.

DOI: 10.1002/smll.201703386

selectable markers such as fluorescent and luminescent proteins have been widely used to track transplanted stem cells over long periods of time;[1–4] however, these markers have multiple limitations for intravital imaging, such as limited depth penetration in tissue, pronounced photobleaching over time, and the presence of autofluorescence in surrounding tissues.[5] In addition, introducing exogenous genes may influence the function and behavior of stem cells.[3]

To circumvent these limitations, a variety of inorganic nanoparticles have been proposed as alternative imaging agents for cell tracking.[1–3] Despite the wide variety of imaging modalities utilized, tracking of stem cells in vivo at a high signaltonoise ratio (SNR) typically requires excess nanoparticle loading. These detection and tracking efforts can influence the stem cell biology or differentiation potential of labeled cells, eventually compromising the outcome of stem cell therapies.[6–8] Surface coatings for enhanced cellular uptake such as cell pen

etrating peptides can elicit membrane perturbations,[9] which may have adverse effects on cellular processes.[10] Excess labeling might also lead to increased degradation of nanoparticles in lysosomes or their efflux from stem cells, ultimately complicating the stem cell tracking analysis.[11]

Given that somatic stem cells are prone to differentiate and lose their multipotency in response to changes in their environment,[12] the load of intracellular nanoparticle label should be effective (i.e., a minimal concentration of intracellular nanoparticles should be employed to prevent adverse effects) without sacrificing the high contrast signal necessary for longterm tracking. Therefore, the design of an appropriate surface chemistry, utilization of high SNR nanoparticles, and optimized conditions for effective cellular labeling with few or negligible negative cellular effects are of particular importance.

Second harmonic generating (SHG) nanocrystals, also referred to as SHG nanoprobes, have recently emerged as versatile and durable labels.[13–16] They can achieve very high SNR due to their very narrow signal spectrum of typically less than 10 nm, permitting single nanoparticle detection and tracking with minimal background signal. SHG nanoprobes are made of noncentrosymmetric crystalline material, whose second

While nanoparticles are an increasingly popular choice for labeling and tracking stem cells in biomedical applications such as cell therapy, their intracellular fate and subsequent effect on stem cell differentiation remain elusive. To establish an effective stem cell labeling strategy, the intracellular nanocrystal concentration should be minimized to avoid adverse effects, without compromising the intensity and persistence of the signal necessary for long-term tracking. Here, the use of second-harmonic generating barium titanate nanocrystals is reported, whose achievable brightness allows for high contrast stem cell labeling with at least one order of magnitude lower intracellular nanocrystals than previously reported. Their long-term photosta-bility enables to investigate quantitatively at the single cell level their cellular fate in hematopoietic stem cells (HSCs) using both multiphoton and electron microscopy. It is found that the concentration of nanocrystals in proliferative multipotent progenitors is over 2.5-fold greater compared to quiescent stem cells; this difference vanishes when HSCs enter a nonquiescent, proliferative state, while their potency remains unaffected. Understanding the nanopar-ticle stem cell interaction allows to establish an effective and safe nanopar-ticle labeling strategy into somatic stem cells that can critically contribute to an understanding of their in vivo therapeutic potential.

Stem Cell Imaging

Successful clinical application of stem cellbased therapies requires the efficient delivery and the appropriate integration and alignment of cells upon transplantation. The development of labeling strategies to track transplanted stem cells promises to advance our understanding of how these cells mediate functional recovery in vivo. Reporter genes that encode for

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harmonic signal is the collective contribution of each asymmetric unit cell. Under intense, pulsed illumination from a multiphoton microscope, SHG nanoprobes effectively combine two identical photons into a single photon of exactly twice the frequency of the incident beam.

This nonabsorptive process comes with several beneficial features for stem cell imaging for future in vivo applications. Unlike commonly used fluorescent probes, SHG nanoprobes neither bleach nor blink, and their signal can become very bright, as it does not saturate with increasing illumination intensity. Since the diameter of the nanoparticles is small with respect to the wavelength of incident light and the wavelength of the SHG signal, multidirectional imaging is possible in deep tissue without sacrificing signal intensity.[14] Combined, these advantages allow SHG nanoprobes to be detected with high sensitivity down to single cell resolution within autofluorescent tissue structures. Previously, SHG nanoprobes have been employed effectively for high speed tracking of cardiac stem cells[17] and deep tissue imaging of skeletal muscle derived stem cells.[18] In order to utilize the advantages of SHG imaging, we set out to assess the capabilities of SHG nanoprobes to suffice the requirement for a bright, robust contrast agent for

effective labeling and tracking of clinically important stem cells. SHG nanoprobes made of tetragonal barium titanate (BaTiO3), which are ferroelectric materials with a perovskite structure, are among the widely used noncentrosymmetric materials due to their high hyperpolarizability[13] and their proven in vivo biocompatibility,[16] whose individual signal can be isolated with high SNR from endogenous SHG.[14]

We reasoned that the bright SHG signal of BaTiO3 would allow us to achieve a satisfactory labeling of stem cells using a small number of nanocrystals, limiting any adverse effect on the wellbeing of the investigated cells. Previously, we employed tetragonal BaTiO3 nanocrystals from commercial sources, typically synthesized by top down approaches such as highenergy ball milling. However, the micrometerrange sizes, heterogeneity, and pronounced aggregation behavior are unsuitable for cell labeling.[16] To produce nanometer sized tetragonal BaTiO3 with defined size distribution and appropriate surface functionalization, we evaluated several production protocols and identified a solvothermal synthesis method that has been shown to produce spherical nanocrystals of

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dynamic light scattering (DLS), transmission electron microscopy (TEM), and Xray diffraction (XRD) (Figure 1b,c). Based on DLS and TEM data, we determined the mean diameter of BaTiO3 nanocrystals produced in this way to be ≈85 nm with a size distribution ranging from 50 to 120 nm (Figure 1b). The XRD spectrum of synthesized BaTiO3 is fitted to a tetragonal phase pattern (Figure 1c).

To accomplish a stable and biocompatible functionalization of BaTiO3 nanocrystals for delivery into stem cells, we followed a stepwise functionalization scheme. First, we coated the hydroxylated surface with positively charged polyllysine (PLL) through electrostatic interactions (Figure 1d and Figure S1a (Supporting Information)). The resulting unbound amine groups on the surface served as a platform for subsequent functionalization. Efficient immobilization of aminereactive fluorescent dyes, which was validated by the high degree of colocalization between fluorescence and the spectrally welldefined SHG signal (Figure S1b, Supporting Information), proved the high conjugation specificity of the PLL coat. The signal colocalization persisted for extended period of time even at low pH values that are encountered in lysosomes (Figure S1c, Supporting Information).[21] In order to minimize the interaction with cellular proteins and to prevent the formation of a protein corona[22,23] around BaTiO3 nanocrystals, which could potentially engage with various biological pathways that might affect the stem cell state, we passivated the nanocrystals with polyethylene glycol (PEG) (Figure 1d and Figure S2a,b,d (Supporting Information)). PEG2000 and PEG5000[24,25] modifications had no noticeable effect on the overall SHG signal intensity (Figure S2c, Supporting Information).

Hematopoietic stem cells (HSCs) constitute a clinically relevant stem cell population that is routinely used in stem cell transplantation to reestablish hematopoietic function in patients whose bone marrow or immune system is compromised. HSCs reside in the bone marrow (BM) cavity in adult mammals and have the unique capacity to replenish the entire blood system[26–28] (Figure 2a), giving rise to a series of multipotent progenitor cells (MPP1–MPP4) with decreasing selfrenewal potential.[29,30] To explore the cellular delivery of SHG nanoprobes into stem cells, we investigated labeling of stem and progenitor populations (i.e., HSC, MPP1, MPP2, and MPP3/4) isolated from BM cells of C57BL/6J mice using fluorescenceactivated cell sorting (FACS) for known unique cell surface proteins (Figure 2b and Figure S3 (Supporting Information)). After isolation, sorted stem cells were incubated for 2 h with equal amounts of uncoated, hydroxylated (BaTiO3OH), PLL treated and PEGylated (BaTiO3PEG2000 and BaTiO3PEG5000) SHG nanoprobes, respectively (Figure 2d). In contrast to PLL coated SHG nanoprobes, PEGylated SHG nanoprobes did not elicit any cytotoxic activity on freshly isolated BM cells (Figure S4, Supporting Information), which deemed the PEG coated SHG nanoprobes suitable for uptake and trafficking studies in stem cells. The intracellular presence of SHG signal per cell was evaluated using nonlinear optical imaging (Figure 2c; Figure S5 and Movies S1 and S2 (Supporting Information)). While BaTiO3OH SHG nanoprobes rarely labeled stem cells, we found that the cellular presence of BaTiO3PEG2000 and – to a lesser degree – BaTiO3PEG5000 SHG nanoprobes was significantly increased for all cells (Figure 2d

and Figure S6 (Supporting Information)). The concentration difference between BaTiO3PEG2000 and BaTiO3PEG5000 SHG nanoprobes is consistent with previous reports on conformational differences between smaller and larger PEG chains and their subsequent protein interactions.[25] Subsequently, we focused our analysis on BaTiO3PEG2000 SHG nanoprobes, as its uptake efficiency was more pronounced.

The electron density of BaTiO3 proved sufficient to directly examine the intracellular presence of SHG nanoprobes at the ultrastructural level using electron microscopy (EM). EM analysis showed that typically one up to a few SHG nanoprobes was present in membrane invaginations, endosomal compartments, and multivesicularlike structures (Figure 2e). To determine which cellular uptake mechanism was responsible for the intracellular delivery of nanoprobes, we investigated the uptake of BaTiO3PEG2000 SHG nanoprobes into HSC and MPP3/4 cells in the presence of various inhibitors that were carefully chosen to block distinct endocytic pathways with minimal crosstalk (Figure S9a,b, Supporting Information). These inhibitors did not elicit any cytotoxic activity on BM derived cells at working concentrations (Figure S7, Supporting Information). Compared to control cells, a pronounced decrease of intracellular SHG signal per cell was noticeable for MPP3/4 cells (reduction up to ≈85%) – and to a lesser extent for HSCs – when cells were treated with chemical inhibitors that targeted the clathrin mediated uptake (Figure S9c, Supporting Information). Filipin, an inhibitor of caveolaeassociated endocytosis, had no significant inhibition effect on the nanoprobe uptake (Figure S9c, Supporting Information). Likewise, cytochalasin D, an actindepolymerizing agent, and nocodazole, a microtubuledisrupting agent, did not affect the presence of intracellular SHG signal, suggesting that actin and microtubuledependent uptake mechanisms such as phagocytosis and macropinocytosis are not committed to the cellular uptake of SHG nanoprobes into HSCs and MPP3/4s (Figure S9c, Supporting Information). These results suggest that BaTiO3PEG2000 SHG nanoprobes enter both HSC and MPP3/4 cell populations mainly through clathrindependent endocytosis.

Although the cellular uptake into HSC and MPP3/4 populations can be regulated by the same endocytic pathway, we noticed that the intracellular presence of BaTiO3PEG2000 SHG nanoprobes in MPP3/4 cells was over 2.5fold greater compared to HSCs (Figure 2c). Since stem cells need to remain intact for the entire life of an organism, they protect themselves from potentially cytotoxic compounds by expressing cell surface adonosine triphosphate (ATP) binding cassette (ABC) pumps, which can actively efflux external substances[31,32] (Figure S9a, Supporting Information). Hence, one possible interpretation is that these pumps actively remove intracellular BaTiO3PEG2000 SHG nanoprobes from HSCs. To address this hypothesis, we blocked the activity of ABC transporters using the chemical inhibitor verapamil, a broadspectrum efflux pump inhibitor,[33] and monitored the effect on the intracellular presence of SHG signal per cell in HSCs and MPP3/4 cells. Flow cytometric analysis demonstrated that treatment with verapamil inhibited the efflux of the DyeCycle Violet dye, leading to the loss of the side population fraction that contains virtually all of HSCs without notable cytotoxicity[32] (Figures S7 and S8, Supporting Information). However, verapamil treatment did not alter the number of

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Figure 2. Cellular delivery of SHG nanoprobes into hematopoietic stem and progenitor cells. a) The mouse hematopoietic system. HSCs give rise to MPPs, which commit to more mature progenitors with restricted cell fate. HSC, hematopoietic stem cells; MPP, multipotent progenitor; CMP, common myeloid progenitor; MEP, megakaryocyte erythroid progenitor; GMP, granulocyte macrophage progenitor; CLP, common lymphoid progenitor; NK, natural killer cell. b) Scheme of isolation and labeling of murine bone-marrow (BM) derived HSCs and MPPs with SHG nanoprobes. c) Confocal images of labeled HSC and MMP3/4 cells showing intracellular presence of SHG nanoprobes. d) Quantitative delivery analysis of SHG nanoprobes into HSCs and MPPs. Freshly isolated cells were incubated with SHG nanoprobes for 2 h at 37 °C. Pooled from three independent experiments. Each dot represents the number of SHG signal per cell, which was segmented using the Spot function of the Imaris image processing program. Mean ± s.d. ****, P < 0.0001, **, P < 0.005, *, P < 0.05 (nonparametric Kruskal–Wallis test with Dunn’s multiple comparison). e) Accompanying electron images showing localization of SHG nanoprobes (arrowheads) in HSC and MPP3/4 cells. Nuc, nucleus.

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intracellular BaTiO3PEG2000 SHG nanoprobes in HSC and MPP3/4 cells (Figure S9d, Supporting Information), suggesting that the ABC transporter activity in HSCs does not account for the difference in cellular delivery of BaTiO3PEG2000 SHG nanoprobes into HSC and MPP3/4.

Since efflux did not seem to be a reason for why HSCs took up fewer BaTiO3PEG2000 SHG nanoprobes than MPP3/4 cells, we explored whether this observation was a consequence of

differential uptake efficiency. Within the BM niche, HSC and MPP3/4 cells have been reported to be in distinct cell cycle states[34,35] (Figure 3a): while the majority of HSCs remain quiescent in the G0 resting phase, ensuring the maintenance of blood homeostasis, MPP3/4 cells are highly proliferative, constantly progressing through the different stages of the cell cycle[29,30,36,37] (Figure 3a and Figure S10 (Supporting Information)). To explore whether the differential cell states influence

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Figure 3. SHG nanoprobes enter efficiently proliferative stem cells and label them stably without significantly affecting stem cell potency. a) Scheme of the cell cycle, which consists of four phases: G1 (interphase: cell size increase), S (DNA synthesis phase), G2 (interphase: preparation for mitosis), M (mitosis). HSCs are considered to be predominantly quiescent (in the G0 phase). Yellow line; restriction point. b) Quantification of anti-Ki67 antibody staining of HSC and MPP3/4 cells freshly isolated (0 h) and incubated for 20 h in growth medium (20 h), respectively. Pooled from three independent experiments (nonparametric Kruskal–Wallis test with Dunn’s multiple comparison). c) Cellular delivery of BaTiO3 SHG nanoprobes into freshly isolated HSC and MPP3/4 cells and into cells that were incubated in growth medium for 20 h, respectively. The cells were incubated with SHG nanoprobes for 2 h. Pooled from three independent experiments, each dot represents the number of SHG signal in a cell. Mean ± s.d. ****, P < 0.0001, ***, P < 0.001, **, P < 0.01, *, P < 0.05 (nonparametric Kruskal–Wallis test with Dunn’s multiple comparison). d) Evaluation of persistence of intracellular SHG nanoprobes for up to 10 h in proliferative MPP3/4 cells. Individual labeled cells were followed by time-lapse imaging. An image was taken every 30 min at 37 °C with 5% CO2 for up to 10 h. The number of intracellular SHG nanoprobes in a cell was quantified at 0, 5, and 10 h. e) Colony-formation unit (%) of multipotent myeloid progenitor cell colonies (GEMM) per multilineage colonies of the labeled and unlabeled cells freshly isolated and cultured in growth medium for 20 h. The cells were incubated with SHG nanoprobes for 45, 90, and 180 min as indicated. G, granulocytes; E, erythrocytes; M, Megakaryocytes; M, macrophage. n = 3, Mean ± s.d.

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uptake of nanoparticles, we compared the level of SHG nanoprobe uptake before and after inducing HSCs to enter the proliferative state. To this end, we incubated HSCs with stem cell factor (SCF) and thrombopoietin (TPO), key growth factors for stem cell selfrenewal[34,35,38] for different time intervals. Then, we monitored the uptake efficiency of pretreated cells by incubating HSCs with BaTiO3PEG2000 SHG nanoprobes for different periods of time (45, 90, and 180 min). We identified 20 h of growth factor treatment followed by at least 90 min SHG nanoprobe incubation as a crucial timeframe to significantly increase nanoparticle uptake (Figure S11, Supporting Information). We confirmed that the intracellular accumulation of BaTiO3PEG2000 SHG nanoprobes did not influence the cell cycle progression of HSC and MPP3/4 cells (Figure S12, Supporting Information). Moreover, the difference in nanoparticle uptake between HSCs and MPP3/4s disappeared after growth factor preincubation (Figure 3b and Figure S11 (Supporting Information)). Using flow cytometry, we confirmed that this treatment period resulted in a major fraction of freshly isolated HSCs, which were initially in the G0/G1 phase, to transition to the proliferative S and G2/M phases (Figure S10, Supporting Information). This outcome was further corroborated when we stained HSC populations with Ki67, a cellular marker for proliferation.[39] Whereas freshly isolated HSCs had very little Ki67, HSCs incubated with growth factor for 20 h displayed increased protein levels, comparable to MPP3/4 cell populations (Figure 3c). Combined, these results illustrate that efficient cellular delivery of BaTiO3PEG2000 SHG nanoprobes takes place when HSCs enter a nonquiescent, proliferative state that seems to favor the energydependent clathrinmediated uptake processes (see Figure S9 in the Supporting Information).

Having established an optimized labeling protocol, we now investigated the intracellular fate of uptaken BaTiO3PEG2000 SHG nanoprobes in individual proliferative MPP3/4 cells over time. Once internalized, nanoparticles typically accumulate over time in acidic intracellular compartments like lysosomes with active degradative enzymes that can lead to loss of signal. To this end, we tracked the SHG nanoprobes over 10 h to monitor changes in SHG signal output over time. The SHG signal per cell remained stable during incubation time, and was split between cells following cell division (Figure 3d and Movies S3 and S4 (Supporting Information)), suggesting that once internalized, SHG nanoprobes persist intracellularly and are not actively exported from labeled cells, consistent with our ABC transporter activity results (see Figure S9 in the Supporting Information). In addition, we evaluated possible adverse effects on the multipotency of HSCs by examining the colonyforming capacity of nonlabeled versus labeled HSCs in vitro. Consistent with a previous report, the colonyforming capacity of proliferative HSCs was modestly reduced.[38] However, no statistically significant differences between the control and labeled cells were detected over SHG nanoprobe incubation time periods that proved optimal for delivery (Figure 3e). Taken together, our approach demonstrates that targeting the proliferative state of cells for optimal labeling times results in an efficient cellular delivery of SHG nanoprobes into somatic stem cells without notable adverse effect on their multipotency.

In summary, we introduce PEGylated SHG nanoprobes as ideal imaging probes for understanding cellular uptake and intracellular fate of nanoparticles in order to achieve improved stem cell labeling. The bright signal of SHG nanoprobes displays longterm photostability even at low pH values, which allows sensitive detection of single intracellular SHG nanoprobes in endolysosomal compartments, without noticeable effects on proliferation and differentiation of multipotent stem cells. The intracellular accumulation of SHG nanoprobes in quiescent and proliferative hematopoietic stem and progenitor cells occurs mainly through clathrinmediated endocytosis. Importantly, inferred from TEM data analysis, high contrast labeling was achieved with at least one order of magnitude less intracellular nanocrystals (less than 100) than previously reported.[40,41] Once internalized, SHG nanoprobes persist without active export and become only diluted in stem cells upon cell division. These results suffice key criteria for potential applications of imaging nanoprobes for the in vivo deeptissue tracking of individual stem cells to study their fate upon transplantation.

By systematically analyzing SHG nanoprobe uptake at the singlecell level of HSCs, we uncovered a critical role of the biological state of adult stem cells in the delivery process. When quiescent HSCs are cultured in growth medium under normoxia conditions, they are primed into proliferation, exiting the quiescent G0 state, which promotes nanoparticle internalization probably through energydependent clathrinmediated processes. Taking advantage of this observation, we adapted a simple yet powerful incubation protocol that yields effective cellular delivery of SHG nanoprobes that can serve as a general guide for enhanced stem cell labeling in order to expand their optical tracking in vivo. Specifically, our approach would allow highly challenging labeling of quiescent stem cell populations by taking advantage of their biology. In addition, we did not observe notable adverse effects on the stem cell multipotency, confirming previous results where sensitive zebrafish embryos injected with SHG nanoprobes developed indistinguishably from their uninjected counterparts.[14] Moreover, our optimized labeling procedure confirms the timesensitive needs in clinical trials, where stem cells are typically transplanted into patients within 24 h after isolation,[1] which will prove critical for the advancement of stem cellbased therapies.

Experimental SectionSynthesis of Barium Titanate Nanocrystals—Synthesis of Bimetallic

Precursor BaTi[OC3H7]6: As previously described,[19] in a N2 glovebox, 5 g of Ba metal (distilled, 99%, Sigma-Aldrich) was added to a 200 mL flask containing 63 mL of anhydrous benzene (99.8%, Sigma), 12 mL of 2-propanol 99.5% (Sigma), and 11 mL of Ti(iPrO)4 (99.99%, Alfa Aesar). The solution was stirred vigorously until the added Ba metal completely dissolved. The solution turned a deep purple color and gradually became milky white after about 5 d. The solution was then placed at 4 °C overnight to help facilitate the precipitation of the precursor, placed in 50 mL centrifuge tubes, and centrifuged, and the pellet twice washed with benzene. After the washing step, the final supernatant was removed and the pellet was allowed to dry in the glovebox for 3 d, yielding a fine powder. This powder was transferred to a sealed glass container for long-term storage.

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Growth of BaTiO3 Nanocrystals: As previously described,[20] acid digestion bombs with 45 mL Teflon liners (Parr) were used for solvothermal reactions. In a typical synthesis, 2.70 g of BaTi[OC3H7]6 was added to the Teflon liner in an O2-free chamber, and 4 mL of N2-purged water was added. The Teflon liner was tightly sealed inside the acid digestion bomb, and the bomb was removed from the chamber and heated in an oven at 220 °C for 18 h. The resulting white nanocrystal precipitate was collected by centrifugation, washed twice with 30 mL of absolute EtOH, and allowed to dry at ambient conditions.

Characterization Using TEM, DLS, and XRD Measurements: TEM images were taken with a FEI Tecnai F30 FEG operating at 300 kV. DLS and zeta potential measurements were taken on a Malvern NanoZS ZetaSizer. BaTiO3 nanocrystal XRD patterns were obtained using a PANalytical X’Pert Pro diffractometer with a Cu Ka source in grazing incidence geometry between 20 and 90 using 50 s per step and a step size of 0.0065.

Functionalization of Barium Titanate Nanocrystals: In order to introduce functional groups on the BaTiO3 surface, 50 mg of nanocrystals was mixed with 10 mL of 1 m nitric acid and was sonicated for 15 min in a bath sonicator. Nanocrystals were precipitated by centrifugation at 3200 × g for 5 min. The supernatant was removed and 5 mL of deionized water was added to the pellet. The nanocrystal suspension was sonicated for 5 min and spun for 5 min at 3200 × g. The supernatant consisted of water dispersible, hydroxyl-functionalized nanocrystals verified by zeta potential measurements and were coated with 70–150 kDa poly-l-lysine (Sigma). Poly-l-lysine (50 mg) was dissolved in 5 mL of 2 × phosphate buffered saline (PBS) (Ambion) and mixed with 5 mL of BaTiO3 nanocrystal suspension, and the mixture was sonicated for 12 h with a probe sonicator. After coating, nanocrystals were centrifuged at 15 000 × g for 5 min in order to remove excess PLL polymers and were washed with 1 × PBS. Resulting BaTiO3 nanocrystals contained free amine groups on their surface that were then further functionalized. Different sizes (2 and 5 kDa) of methoxypolyethylene glycol acetic acid N-succinimidyl esters (Sigma) were dissolved in anhydrous dimethyl sulfoxide, as 500 mg mL−1 stock solutions. In order to fully cover the nanocrystal surface, 200 µL of PLL coated BaTiO3 nanocrystals were incubated with 2 and 5 kDa PEG succinimidyl ester solutions with a final PEG concentration of 5 × 10−3 m. In a similar setup, the amine-reactive PEG concentration varied in order to generate nanocrystals with different surface charges. The nanocrystal suspension was then washed twice in 1 × PBS through centrifugation at 15 000 × g for 5 min. Surface charges of these samples were characterized using zeta potential measurements.

Assessing the Stability of BaTiO3 SHG Nanoprobe Functionalization: In order to confirm the functionalization efficiency, 1 mg of Alexa Fluor 488 succinimidyl ester (LifeTech) was dissolved in 1 mL of anhydrous dimethylformamide (Sigma). An aliquot of 0.3 µL dye solution was added to 200 µL of PLL-coated nanocrystals and incubated for 2 h. The nanocrystal suspension was then washed twice in 1 × PBS through centrifugation at 15 000 × g for 5 min. In order to assess the pH stability of BaTiO3 SHG nanoprobe coating, they were incubated in Tris-HCl solutions with pH values ranging from 4 to 7. Alexa 488 conjugated SHG nanoprobes were incubated in these solutions for 72 h and washed afterward twice with 1 × PBS before imaging.

Imaging of BaTiO3 Nanocrystals: In order to immobilize BaTiO3 SHG nanoprobes, they were embedded in a polyacrylamide gel by mixing 112.5 µL Tris (Sigma) (1 m, pH 7), 3 µL of 5 mg mL−1 Alexa 488 conjugated nanoparticle suspension, 180 µL 30% acrylamide/bis-acrylamide (37:1 ratio) (Sigma), 1.5 µL 10% ammonium persulfate (Sigma), and 0.3 µL tetramethylethylenediamine (TEMED) (Sigma), which cross-linked the gel within 20 min, inside 8-well imaging chambers (Lab-Tek). Imaging experiments were performed on a Zeiss LSM 780 microscope (Carl Zeiss AG) with the LD LCI Plan-Apochromat 25×/0.8 water objective lens (Carl Zeiss AG) equipped with a spectral GaAsP detector and a tunable two-photon laser source (Chameleon Ultra II, Coherent Inc.). During the imaging experiments, SHG nanoprobes were illuminated with 850 nm incident wavelength and the corresponding SHG signal was detected with multispectral detection in Lambda mode of ZEN software ranging from 412 to 595 nm with 8.8 nm width in each channel. The Manders’

overlap coefficient was used to quantify the degree of colocalization between the Alexa Fluor 488 and the SHG nanoprobe signals.

Mouse Work: 8–14 week-old C57Bl/6 mice were purchased from Charles River (France). Mice were maintained in the ETH Zurich D-BSSE animal facility under specific pathogen free conditions in individually ventilated cages. All the animal procedures were performed according to the Swiss Cantonal Veterinary Office regulations (authorization number 2563).

Isolation and Expansion of Hematopoietic Stem Cell Populations: BM was isolated from mouse tibiae, femora, and coxal bones by crushing them in PBS containing 2 × 10−3 m ethylenediaminetetraacetic acid (EDTA) and 5% bovine serum albumin (BSA), filtrated through a 70 µm cell strainer. Lineage negative BM cells were prepared by labeling the BM cell suspension with a mixture of purified biotinylated monoclonal antibodies CD3 (145-2C11), CD11b (M1/70), CD45R (RA3-6B2), Ly-6G (RB6-8C5), TER-119 (TER-119) (eBioscience), and CD19 (6D5) (BioLegend). Lineage positive cells were removed by two to three rounds of magnetic bead depletion with streptavidin-conjugated beads (eBioscience). Cells were then stained with six fluorophore-conjugated antibodies; streptavidin-conjugated Brilliant Violet 421, CD150 (TC15-12F12.2) PE, Sca-1 (9E13-161.7) APC-Cy7 (BioLegend), c-Kit (2B8) PE-Cy7, CD48 (HM48-1) APC, and CD34 (RAM34) fluorescein isothiocyanate (FITC) (eBioscience). FACS was performed using a FACS Aria (Becton Dickinson). HSC, MPP1, MPP2, and MPP3/4 were gated with Linneg, Sca-1+, c-Kit+ (LSK), CD34−, CD150+, CD48−; LSK, CD34+, CD150+, CD48−; LSK, CD34+, CD150+, CD48+; LSK, CD34+, CD150−, CD48+, respectively (see Figure S3 in the Supporting Information). Dead cells were gated out using 4′,6 diamidino-2-phenylindole (DAPI) staining. Data were analyzed using FlowJo software (Tree Star). Freshly sorted hematopoietic stem and progenitor cell populations were cultured in a 96-well rounded bottom plate using StemSpan serum-free medium (STEMCELL Technologies) supplemented with 50 ng mL−1 of murine recombinant SCF and TPO (PeproTech EC Ltd) at 37 °C in an atmosphere of >95% humidity and 5% CO2.

Cell Labeling with SHG Nanoprobes: Freshly isolated or preincubated cells were incubated with SHG nanoprobes at 10 µg mL−1 for 2 h in a 96-well rounded bottom plate. For the inhibitor assays, cells were incubated with respective inhibitors for 15 min and during the 2 h incubation with SHG nanoprobes. After labeling, cells were washed twice with 3 mL PBS and centrifuged at 250 g for 3 min to form a pellet. Supernatant was removed and the cells were suspended with supplemented StemSpan serum-free medium. For quantitative analysis of intracellular signal of SHG nanoprobes, labeled cells were seeded on 12-well adhesion slides (Marienfeld) and incubated for 1 h in a cell culture incubator at 37 °C, >95% humidity, 5% CO2. The cells were then fixed with 4% paraformaldehyde (PFA) and stained with CellMask Orange Plasma membrane Stain (Life Technologies) and DAPI for nucleus. The slides were mounted with #1.5 coverglass using Vectashield antifading reagent (Vector Laboratories). Confocal images were obtained using a Zeiss LSM 780 microscope (Carl Zeiss AG) with a Plan-Apochromat 63×/1.4 oil objective lens (Carl Zeiss AG) equipped with a spectral GaAsP detector and a tunable two-photon laser source (Chameleon Ultra II, Coherent Inc.). Time-lapse imaging was performed on a Zeiss LSM 780 microscope (Carl Zeiss AG) with a C-Apochromat 40×/1.1 water objective lens (Carl Zeiss AG) equipped with a spectral GaAsP detector and a tunable two-photon laser source (Chameleon Ultra II, Coherent Inc.). The MPP3/4 cells labeled with SHG nanoprobes were seeded on μDish35mm 4-well plate (Ibidi). An image was taken every 30 min at 37 °C with 5% CO2 for up to 10 h. All movies were compiled and the change in the number of intracellular SHG nanoprobes was quantitatively monitored using the IMARIS software using a custom MATLAB script (Movies S3 and S4, Supporting Information).

Cytotoxicity Assay: In order to test the cytotoxicity of SHG nanoprobes and chemicals, a lactate dehydrogenase (LDH) cytotoxicity assay was performed according to the manufacture’s protocol (Roche). The cells were incubated with respective SHG nanoprobes or chemicals in 150 µL of supplemented Stemspan media for indicated periods of time in a 96-well round bottom plate. 10% Triton-X was used as positive control that induces 100% cell lysis. The plate was centrifuged at 250 g for

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5 min, and 100 µL of supernatant was collected and mixed with 100 µL of detection reagent mix in a new 96-well plate. After 30 min incubation at room temperature in dark, the absorbance was read at 490 and 600 nm reference wavelengths using M1000 Pro Reader (Tecan) to measure the relative amount of LDH in the media released from the damaged cells.

Electron Microscopy: HSCs were fixed in 2% paraformaldehyde (Sigma) and 2.5% glutaraldehyde (Sigma) in 0.15 m sodium cacodylate (Sigma) buffer, pH 7.4 for 15 min. Cells were then centrifuged using Beckman Microfuge B (Beckman) at 500 rfc for 5 min to form a pellet. The supernatant was removed and the cells were taken up into cellulose tubes and fixed with fresh fixative buffer for 1 h. The samples were washed three times with deionized water and postfixed in 2% aqueous osmium tetroxide (Sigma) for 1 h. Later, they were washed again with deionized water and were incubated in 1% aqueous uranyl acetate overnight for staining. The samples were dehydrated in a graded series of ethanol (50%, 70%, 90%, and 3 times 100% for 20 min each) and anhydrous acetone (Sigma) for 1 h. They were then infiltrated with Epon (Fluka Epon Kit, standard recipe) in acetone (25% and 50% for 2 h each, 75% overnight, and 3 times 100% Epon), before polymerizing at 60 °C for 3 d. 50 nm sections were cut using a diamond knife (Diatome) in an ultramicrotome (Leica), stained with uranyl acetate and lead citrate, and imaged in a transmission electron microscope (FEI Morgagni 268). The regular size and spherical shape of the electron dense spots in the acquired EM pictures of adult stem cells, which were previously incubated with SHG nanoprobes, indicate that they do not designate cellular structures, but SHG nanoprobes.

Side-Population Analysis Using Flow Cytometry: BM derived cells were incubated at 37 °C for 90 min with 10 × 10−6 m Vybrant DyeCycle Violet (Life Technologies). For the ABC transporter inhibitor assay, the cells were incubated with 50 × 10−6 m verapamil for 15 min and during the 90 min incubation with DyeCycle. After incubation, lineage positive cells were removed as described above, and lineage negative BM cells were stained with Sca-1 (9E13-161.7) APC-Cy7 (BioLegend) and c-Kit (2B8) PE-Cy7 for LSK gating. Side-population analysis was performed using LSRFortessa (Becton Dickinson). Dead cells were gated out using DAPI staining. DyeCycle was excited with 405 nm illumination and fluorescence emission was measured with blue band-pass (450/50) and red band-pass (675/20) optical filters. Data were analyzed using the FlowJo software (Tree Star).

Ki67 Staining of Cell Populations: Freshly isolated or preincubated cells were incubated with SHG nanoprobes at 10 µg mL−1 for 2 h in a 96-well rounded bottom plate. Cells were centrifuged and immobilized on 12-well adhesion slides as previously described. The cells were then fixed with 4% PFA and permeabilized with 0.1% Triton X in 5% BSA for 30 min. The cells were then incubated with rabbit polyclonal anti Ki67 antibody for 1 h (Abcam) at 1:100 dilution and washed three times with PBS. Cells were then incubated with the secondary antibody, anti-rabbit Alexa 488, and CellMask Orange Plasma Membrane Stain for 30 min and washed three times with PBS. The cells were then mounted using VectaShield along with DAPI as a nucleus marker. Images were taken with a 20× Zeiss objective with 0.8 numerical aperture (NA). The DAPI channel was used as mask, and intensity values of each Ki67 stained nucleus were measured using ImageJ.

Cell Cycle Analysis Using Flow Cytometry: EdU labeling and cell cycle analysis were performed using the Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit according to the manufacture’s protocol (Life technologies). Freshly sorted and preincubated HSC and MPP3/4 cells (minimum 300 cells) were seeded in a 96-well round bottom plate. The cells were incubated with or without 10 µg mL−1 of SHG nanoprobes in the medium containing 20 × 10−6 m of EdU for 2 h. EdU-treated cells were fixed with PFA-based fixative provided with the assay kit. Cells were then stained with Alexa Fluor 488 dye azide for detection of newly synthesized DNA, and total DNA was stained with 0.5 µg mL−1 DAPI. Cell cycle analysis was performed using LSRFortessa (Becton Dickinson), and the cell cycle state was analyzed using the FlowJo software (Tree Star).

Colony-Formation Assay: The methylcellulose colony-forming assay was performed using MethoCult GF M3434 (STEMCELL Technologies). For triplicate analyses, 200 HSC or MPP cells were seeded in a

well of a 96-well round bottom plate and incubated without or with SHG nanoprobes for indicated periods of time. After labeling, the cells were washed twice with 3 mL PBS and centrifuged at 250 g for 3 min. Supernatant was removed and cells were suspended in 8 mL of MethoCult, and 2 mL of cell suspension was plated into a 35 mm plate and allowed to grow for 12 d. The colonies generated in MethoCult were scored as GEMM or other type by monitoring size and morphology of each colony under light microscope using an MVX10 Research Macro Zoom Microscope (OLYMPUS). The colonies were also subjected to cytospin and characterized morphologically and cytochemically by Hemacolor Rapid staining of blood smear (Merck Millipore) using the MVX10 light microscope (OLYMPUS).

Image Analysis: Intracellular SHG nanoprobes were quantified by segmenting independent SHG positive regions as individual endosomes using Imaris’ Spots function along with a custom MATLAB script (The Mathworks, Inc. Natick, Ma). The single-photon confocal images shown in Figure 2c; Figure S5 and Movies S1 and S2 (Supporting Information) were denconvolved by the Huygens software (Scientific Volume Imaging, Hilversum, NL). The SHG channel obtained by two-photon imaging was filtered with a 5 × 5 × 5-voxel median filter to remove high-frequency background noise.

Statistical Analysis: All numerical values represent mean ± s.d. Statistical significance was determined using nonparametric Kruskal–Wallis test and Dunn’s multiple comparison test to analyze mean rank difference.

Supporting InformationSupporting Information is available from the Wiley Online Library or from the author.

AcknowledgementsN.S. and A.Y.S. contributed equally to this work. A.Y.S. and B.E.C. synthesized BaTiO3 nanocrystals; A.Y.S. generated all modifications and performed all analyses of BaTiO3 SHG nanoprobes; N.S. designed all HSC experiments and analyzed the data with the help of A.Y.S. and R.T.; N.S., A.Y.S., and P.P. wrote the paper, with contributions from B.E.C. and R.T. P.P. supervised the work. The authors thank all members of the Pantazis Lab for discussion and advice. The authors thank M. A. Mohr, W. P. Dempsey, and M. Welling for comments on the paper. The authors would like to thank the Single Cell Facility (SCF) at the Department of Biosystems Science and Engineering (D-BSSE) for technical support, especially V. Jäggin and T. Lopes for excellent technical assistance for the FACS and the flow cytometric analyses, and A. Ponti for image processing and for supporting the analysis with a custom MATLAB script. The authors also thank A. Alitalo, H. Oller, G. Camenisch, and D. Zimmer for animal husbandry at the ETH Zurich D-BSSE animal facility. The authors thank P. Hoppe from the group of Prof. T. Schröder (ETH Zurich), T. Shimizu from the group of Prof. R. Skoda (University of Basel), and G. Nusspaumer from the group of Prof. R. Zeller (University of Basel) for advice on hematopoietic cell sorting, culture, and experiments. The authors also thank Prof. M. Niederberger (ETH Zurich) and his group for their help in obtaining X-ray diffraction data. The authors thank the Biophysics Facility of the Biozentrum (University of Basel) for providing access to their NanoZS ZetaSizer instrument. The authors kindly acknowledge the Scientific Center for Optical and Electron Microscopy (ScopeM) for their help in imaging BaTiO3 nanocrystals. Work at the Molecular Foundry was supported by the Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. This work was supported by the Swiss National Science Foundation (SNF grant no. 31003A_144048), the European Union Seventh Framework Program (Marie Curie Career Integration Grant (CIG) no. 334552), and the Swiss National Center of Competence in Research (NCCR) “Nanoscale Science.”

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Conflict of InterestThe authors declare competing financial interests. Aspects of the work mentioned in this work are the subject of granted patents and patent applications filed by the Swiss Federal Institute of Technology in Zurich (ETH Zurich), Zurich, Switzerland and the California Institute of Technology (Caltech), Pasadena, CA, USA.

Keywordsbarium titanate, cell cycle, hematopoietic stem cells, second harmonic generation

Received: September 28, 2017Revised: November 14, 2017

Published online:

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