Development of single-cell Raman spectroscopy for noninvasively screening the differentiation of living stem and progenitor cells in biomaterial platforms for 2D and 3D cell culture

Stem and progenitor cells' capabilities to self-renew, differentiate, and proliferate within their native tissue made them the subject of ongoing studies to understand the mechanisms of such processes and their potential use as therapeutics. Regenerative medicine encompasses the idea of treating malignant diseases and disorders by replacing mutated cells with healthy stem cells transplanted into a patient for the return of the normal function of tissues or organs. The translation of in vitro and in vivo studies of stem cell delivery to disease tissues for regeneration has been hindered by the limited survival of the transplanted stem cells, loss of potency and quality, and the inability of the cells to migrate to target sites. An example of limited translation of stem cell treatments to the clinic is the fact that after more than five decades of research, only hematopoietic stem cells (HSCs) are approved by the Food and Drug Administration as a stem cell treatment. To solve the challenges involving the transplantation of stem cells for the treatment of malignant diseases, biomaterials have been studied for the optimization of delivery and potency of stem cell therapies. The idea is to incorporate a biomaterial that is bio-compatible and bio-degradable to facilitate the delivery of stem cells into damaged tissues with improved retention. Hydrogels are one of the most studied biomaterials for this purpose. They can mimic the natural extracellular matrix (ECM) to provide a protective barrier as the cells migrate to target sites, avoiding harsh conditions during transport. To develop stem cell therapies that efficiently and safely deliver stem cells to malignant tissues using hydrogels, the cell responses within them need extensive characterization. These include real-time tracking of migration to disease sites and release of the stem cell from the hydrogel. The characterization of these mechanisms relies on fluorescent markers, stimulated emission depletion imaging, PET scans, and radio labeling. One of the main limitations of these characterization methods is the lack of sustainable detection and fast self-degradation of labeling materials. Motivated by the efforts on optimizing and expanding the use of HSCs in the treatment of leukemias and other blood and immune diseases we focused most of the work presented here on HSCs and other hematopoietic populations with differentiation potential. Because the combinations of extrinsic cues within the HSC niche that lead to HSCs fate decisions remain unknown, there is still a need for noninvasive and location-specific techniques to enable the identification of the differentiation stages of individual hematopoietic cells on biomaterial platforms for 2D and 3D cell culture applications. The work presented here uses Raman microspectroscopy to analyze the differentiation stages of single stem and progenitor cells in 2D and 3D cell cultures. This approach needed the development of a custom Raman-compatible substrate, presented in Chapter 2, to be used as an inexpensive substrate for constructing biomaterial platforms that enable the use of Raman microspectroscopy to non-invasively identify the fate decisions of stem cells in response to extrinsic cues. Here we showed in Chapter 3 that a partial-least squares discriminant analysis (PLS-DA) model of single-cell Raman spectra enabled 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 (CMP), and common lymphoid progenitor (CLP) on top of functionalized polyacrylamide hydrogels with varying stiffness. In Chapter 4 we showed the feasibility of identifying the fate decision of the hematopoietic cell line, THP-1 cells, within collagen and gelatin methacrylate hydrogels by accurate classification of the three differentiated populations using PLS-DA of their Raman spectra. This work serves as a proof-of-concept for the development of a noninvasive tool that identifies stem cell fate decisions in biomaterials with location-specificity. These capabilities should be further optimized for the analysis of controlled microarrays that mimics complex ECM to ensure the robustness of the Raman spectral signatures from cells used for fate identification.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemical Engineering

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2022

Author dc:creator

• Pastrana-Otero, Isamar

Contributors dc:contributor

• Kraft, Mary L.
• Harley, Brendan A.C.
• Underhill, Gregory H.
• Kong, Hyunjoon

• Raman Spectroscopy
• Stem Cells
• Multivariate Analysis
• 3d Cell Culture
• Hematopoietic Stem Cells
• Stem Cell Microenvironment
• Biomaterial Platforms
• Hydrogels

Statement dc:rights

• © 2022 Isamar Pastrana-Otero

Language dc:language

eng

Handle dc:identifier

https://hdl.handle.net/2142/117681

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/117681