Single-cell analysis of hematopoietic cell fate on niche-mimetic platforms using Raman microspectroscopy

Regenerative therapies are predicated on our ability to achieve controlled and reproducible expansion of stem cells ex vivo. Hematopoietic stem cells (HSCs) are the best-studied class of tissue-restricted stem cells and their microenvironment in vivo has also been the subject of considerable research. Although a host of cellular, molecular and physical cues that regulate stem cell fate in vivo have been identified, efforts to culture HSCs for extended periods by recapitulating some of these interactions in vitro have met with limited success. Miniaturized biomaterial platforms that present tens to hundreds of putative niche components can help screen individual factors or their combinations which drive quiescence, self-renewal and differentiation in vitro while reducing the numbers of rare cell populations required for testing. Screening approaches rely on accurate identification of specific stem cell outcomes in order to correlate extrinsic cues with the fates they elicit. Traditionally, HSCs and their progeny populations have been identified using time-consuming functional assays or immunofluorescence detection of a combination of surface antigens, both of which are invasive and preclude longitudinal analysis of cell fate in culture. Methods that allow rapid, label-free and location-specific characterization of the differentiation stages of individual cells would enable the rapid development of concurrent screens of the effects of niche components and their combinations on stem cell fate specification. Raman spectroscopy has emerged as one of the few analytical approaches that can be used to noninvasively assay the biomolecular composition of cells. Combining Raman spectroscopy with multivariate methods such as partial least-squares discriminant analysis and self-organizing maps may enable objective classification of specific stem cell outcomes on screening platforms, or facilitate investigations into the clonal heterogeneity of stem cells, respectively. The work presented herein describes the use of spontaneous Raman microspectroscopy and multivariate methods as a noninvasive technique for following hematopoietic stem cells and their progeny in vitro. Chapter 1 introduces the field of tissue engineering and the role of the stem cell microenvironment in guiding stem cell fate especially as it relates to the hematopoietic stem cell and lays down the background and motivation for label-free analytical approaches for phenotypic analysis of stem cells in vitro. Chapter 2 discusses the development of a hydrogel culture platform that is compatible with Raman spectroscopy, and demonstrates the capability of multivariate methods to capture subtle changes in the biochemical make-up of cells using mammalian cell lines. Chapter 3 uses a monocytic leukemia cell line capable of differentiating into macrophages (THP-1 line) to build on the work presented in Chapter 2 and provide proof of concept for the feasibility of real-time tracking of cell differentiation using Raman spectroscopy. This chapter introduces self-organizing maps (SOMs). Finally, Chapter 4 shows the successful use of Raman spectroscopy and partial least-squares discriminant analysis for determining the differentiation stages of individual living hematopoietic stem cells and their progeny on biomaterial substrates with varying stiffness. Chapter 5 presents a short discussion of the implications of this work for the field and summarizes some of the issues and challenges that must be addressed in future work using Raman spectroscopy on HSCs.

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

2021

Author dc:creator

• Majumdar, Sayani

Contributors dc:contributor

• Kraft, Mary L
• Harley, Brendan A
• Kong, Hyun Joon
• Underhill, Gregory H

• Raman microspectroscopy
• Multivariate analysis
• Cell discrimination
• Single-cell Raman spectra
• Hematopoietic stem cells
• Stem cell differentiation
• Noninvasive phenotypic analysis
• Stem cell niche

Statement dc:rights

• Copyright 2021 Sayani Majumdar

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/110709

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/110709