Stanford Advisors

All Publications

  • Identification of the Differentiation Stages of Living Cells from the Six Most Immature Murine Hematopoietic Cell Populations by Multivariate Analysis of Single-Cell Raman Spectra. Analytical chemistry Pastrana-Otero, I., Majumdar, S., Gilchrist, A. E., Harley, B. A., Kraft, M. L. 2022


    Efforts to expand hematopoietic stem and progenitor cells (HSPCs) in vitro are motivated by their use in the treatment of leukemias and other blood and immune system diseases. The combinations of extrinsic cues within the hematopoietic stem cell (HSC) niche that lead to HSC fate decisions remain unknown. New noninvasive and location-specific techniques are needed to enable identification of the differentiation stages of individual hematopoietic cells on biomaterial microarray screening platforms that minimize the usage of rare HSCs. Here, we show that a combination of Raman microspectroscopy and partial least-squares discriminant analysis (PLS-DA) enables the location-specific identification of individual living cells from the six most immature hematopoietic cell populations, HSC, multipotent progenitor (MPP)-1, MPP-2, MPP-3, common myeloid progenitor, and common lymphoid progenitor. Better than 90% accuracy was achieved. We show that the accuracy of this differentiation stage identification was based on spectral features associated with cell biochemistries. This work establishes that PLS-DA can capture the subtle spectral variations between as many as six closely related cell populations in the presence of potentially significant within-population spectral variation. This noninvasive approach can be used to screen HSC fate decisions elicited by extrinsic cues within biomaterial microarray screening platforms.

    View details for DOI 10.1021/acs.analchem.2c00714

    View details for PubMedID 36001072

  • Engineered tissue models to replicate dynamic interactions within the hematopoietic stem cell niche. Advanced healthcare materials Gilchrist, A. E., Harley, B. A. 1800: e2102130


    Hematopoietic stem cells are the progenitors of the blood and immune system and represent the most widely used regenerative therapy. However, their rarity and limited donor base necessitates the design of ex vivo systems that support HSC expansion without the loss of long-term stem cell activity. This review describes recent advances in biomaterials systems to replicate features of the hematopoietic niche. Inspired by the native bone marrow, these instructive biomaterials provide stimuli and cues from co-cultured niche-associated cells to support HSC encapsulation and expansion. Engineered systems increasingly enable study of the dynamic nature of the matrix and biomolecular environment as well as the role of cell-cell signaling (e.g., autocrine feedback versus paracrine signaling between dissimilar cells). The inherent coupling of material properties, biotransport of cell-secreted factors, and cell-mediated remodeling motivate dynamic biomaterial systems as well as characterization and modeling tools capable of evaluating a temporally evolving tissue microenvironment. Recent advances in HSC identification and tracking, model-based experimental design, and single-cell culture platforms facilitate the study of the effect of constellations of matrix, cell, and soluble factor signals on HSC fate. While inspired by the HSC niche, these tools are amenable to the broader stem cell engineering community. This article is protected by copyright. All rights reserved.

    View details for DOI 10.1002/adhm.202102130

    View details for PubMedID 34936239

  • Precise control of synthetic hydrogel network structure via linear, independent synthesis-swelling relationships SCIENCE ADVANCES Richbourg, N. R., Wancura, M., Gilchrist, A. E., Toubbeh, S., Harley, B. C., Cosgriff-Hernandez, E., Peppas, N. A. 2021; 7 (7)


    Hydrogel physical properties are tuned by altering synthesis conditions such as initial polymer concentration and polymer-cross-linker stoichiometric ratios. Traditionally, differences in hydrogel synthesis schemes, such as end-linked poly(ethylene glycol) diacrylate hydrogels and cross-linked poly(vinyl alcohol) hydrogels, limit structural comparison between hydrogels. In this study, we use generalized synthesis variables for hydrogels that emphasize how changes in formulation affect the resulting network structure. We identify two independent linear correlations between these synthesis variables and swelling behavior. Analysis through recently updated swollen polymer network models suggests that synthesis-swelling correlations can be used to make a priori predictions of the stiffness and solute diffusivity characteristics of synthetic hydrogels. The same experiments and analyses performed on methacrylamide-modified gelatin hydrogels demonstrate that complex biopolymer structures disrupt the linear synthesis-swelling correlations. These studies provide insight into the control of hydrogel physical properties through structural design and can be used to implement and optimize biomedically relevant hydrogels.

    View details for DOI 10.1126/sciadv.abe3245

    View details for Web of Science ID 000617708700026

    View details for PubMedID 33579714

    View details for PubMedCentralID PMC7880590

  • Development of an inexpensive Raman-compatible substrate for the construction of a microarray screening platform ANALYST Pastrana-Otero, I., Majumdar, S., Gilchrist, A. E., Gorman, B. L., Harley, B. C., Kraft, M. L. 2020; 145 (21): 7030-7039


    Biomaterial microarrays are being developed to facilitate identifying the extrinsic cues that elicit stem cell fate decisions to self-renew, differentiate and remain quiescent. Raman microspectroscopy, often combined with multivariate analysis techniques such as partial least square-discriminant analysis (PLS-DA), could enable the non-invasive identification of stem cell fate decisions made in response to extrinsic cues presented at specific locations on these microarrays. Because existing biomaterial microarrays are not compatible with Raman microspectroscopy, here, we develop an inexpensive substrate that is compatible with both single-cell Raman spectroscopy and the chemistries that are often used for biomaterial microarray fabrication. Standard deposition techniques were used to fabricate a custom Raman-compatible substrate that supports microarray construction. We validated that spectra from living cells on functionalized polyacrylamide (PA) gels attached to the custom Raman-compatible substrate are comparable to spectra acquired from a more expensive commercially available substrate. We also showed that the spectra acquired from individual living cells on functionalized PA gels attached to our custom substrates were of sufficient quality to enable accurate identification of cell phenotypes using PLS-DA models of the cell spectra. We demonstrated this by using cells from laboratory lines (CHO and transfected CHO cells) as well as adult stem cells that were freshly isolated from mice (long-term and short-term hematopoietic stem cells). The custom Raman-compatible substrate reported herein may be used as an inexpensive substrate for constructing biomaterial microarrays that enable the use of Raman microspectroscopy to non-invasively identify the fate decisions of stem cells in response to extrinsic cues.

    View details for DOI 10.1039/d0an01153c

    View details for Web of Science ID 000582413100027

    View details for PubMedID 33103665

    View details for PubMedCentralID PMC7594104

  • Connecting secretome to hematopoietic stem cell phenotype shifts in an engineered bone marrow niche INTEGRATIVE BIOLOGY Gilchrist, A. E., Harley, B. C. 2020; 12 (7): 175-187


    Hematopoietic stem cells (HSCs) primarily reside in the bone marrow, where they receive external cues from their local microenvironment. The complex milieu of biophysical cues, cellular components and cell-secreted factors regulates the process by which HSC produce the blood and immune system. We previously showed direct coculture of primary murine hematopoietic stem and progenitor cells with a population of marrow-derived mesenchymal stromal and progenitor cells (MSPCs) in a methacrylamide-functionalized gelatin (GelMA) hydrogel improves hematopoietic progenitor maintenance. However, the mechanism by which MSPCs influenced HSC fate decisions remained unknown. Herein, we report the use of proteomic analysis to correlate HSC phenotype to a broad candidate pool of 200 soluble factors produced by combined mesenchymal and hematopoietic progeny. Partial least squares regression (PLSR), along with an iterative filter method, identified TGFβ-1, MMP-3, c-RP and TROY as positively correlated with HSC maintenance. Experimentally, we then observe exogenous stimulation of HSC monocultures in GelMA hydrogels with these combined cytokines increases the ratio of hematopoietic progenitors to committed progeny after a 7-day culture 7.52 ± 3.65-fold compared to non-stimulated monocultures. Findings suggest a cocktail of the downselected cytokines amplifies hematopoietic maintenance potential of HSCs beyond that of MSPC-secreted factors alone. This work integrates empirical and computation methods to identify cytokine combinations to improve HSC maintenance within an engineered HSC niche, suggesting a route toward identifying feeder-free culture platforms for HSC expansion. Insight Hematopoietic stem cells within an artificial niche receive maintenance cues in the form of soluble factors from hematopoietic and mesenchymal progeny. Applying a proteomic regression analysis, we identify a reduced set of soluble factors correlated to maintenance of a hematopoietic phenotype during culture in a biomaterial model of the bone marrow niche. We identify a minimum factor cocktail that promotes hematopoietic maintenance potential in a gelatin-based culture, regardless of the presence of mesenchymal feeder cells. By combining empirical and computational methods, we report an experimentally feasible number of factors from a large dataset, enabling exogenous integration of soluble factors into an engineered hematopoietic stem cell for enhanced maintenance potential of a quiescent stem cell population.

    View details for DOI 10.1093/intbio/zyaa013

    View details for Web of Science ID 000595647400001

    View details for PubMedID 32556172

    View details for PubMedCentralID PMC7384206

  • Soluble Signals and Remodeling in a Synthetic Gelatin-Based Hematopoietic Stem Cell Niche ADVANCED HEALTHCARE MATERIALS Gilchrist, A. E., Lee, S., Hu, Y., Harley, B. C. 2019; 8 (20): e1900751


    Hematopoietic stem cells (HSCs) reside in the bone marrow within niches that provide microenvironmental signals in the form of biophysical cues, bound and diffusible biomolecules, and heterotypic cell-cell interactions that influence HSC fate decisions. This study seeks to inform the development of a synthetic culture platform that promotes ex vivo HSC expansion without exhaustion. A library of methacrylamide-functionalized gelatin (GelMA) hydrogels is used to explore remodeling and crosstalk from mesenchymal stromal cells (MSCs) on the expansion and quiescence of murine HSCs. The use of a degradable GelMA hydrogel enables MSC-mediated remodeling, yielding dynamic shifts in the matrix environment over time. An initially low-diffusivity hydrogel for co-culture of hematopoietic stem and progenitor cells to MSCs facilitates maintenance of an early progenitor cell population over 7 days. Excitingly, this platform promotes retention of a quiescent HSC population compared to HSC monocultures. These studies reveal MSC-density-dependent upregulation of MMP-9 and changes in hydrogel mechanical properties (ΔE = 2.61 ± 0.72) suggesting MSC-mediated matrix remodeling may contribute to a dynamic culture environment. Herein, a 3D hydrogel is reported for ex vivo HSC culture, in which HSC expansion and quiescence is sensitive to hydrogel properties, MSC co-culture, and MSC-mediated hydrogel remodeling.

    View details for DOI 10.1002/adhm.201900751

    View details for Web of Science ID 000486917700001

    View details for PubMedID 31532901

    View details for PubMedCentralID PMC6813872