Methods to assess stem cell lineage, fate and function

Abstract

Stem cell therapy has the potential to regenerate injured tissue. For stem cells to achieve their full therapeutic potential, stem cells must differentiate into the target cell, reach the site of injury, survive, and engraft. To fully characterize these cells, evaluation of cell morphology, lineage specific markers, cell specific function, and gene expression must be performed. To monitor survival and engraftment, cell fate imaging is vital. Only then can organ specific function be evaluated to determine the effectiveness of therapy. In this review, we will discuss methods for evaluating the function of transplanted cells for restoring the heart, nervous system, and pancreas. We will also highlight the specific challenges facing these potential therapeutic areas.

Figure 1

Schematic of the key steps in evaluating the lineage, fate, and function of transplanted stem cells for the treatment of cardiovascular disease. In this schematic, human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) undergo differentiation into cardiomyocytes in vitro prior to injection into the mouse heart. Abbreviations: MRI, magnetic resonance imaging

Figure 2

Schematic for non-invasive imaging of stem cell fate in the myocardium using iron particle labeling, radionuclide labeling and reporter gene labeling. Abbreviations: SPIO, superparamagnetic iron oxide; ¹⁸F-FDG, 18F-fluorodeoxyglucose; ^99mTc, 99mTechnetium; ¹¹¹ln-Oxine, 111 Indium-Oxine.

Figure 3

Tracking of transplanted stem cells by iron particle labeling with histological confirmation in an experimental stroke model. (a-c) Horizontal and (d-f) frontal MRI scans show dose-dependent size of iron oxide labeled grafts as hypointense arrows in the striatum (arrow) medially in the penumbral zone of the stroke, which appears as strongly hyperintense areas on T2-weighted images. The cell doses are as follows: (a,d) 50,000 cells (low dose), (b,e) 200,000 cells (intermediate dose), and (c,f) 400,000 cells (high dose). (g-i) Three dimensional surface rendering reconstruction of high resolution T2-weighted MRI of brain shows grafts in green and stroke in pink and red from the low dose (g-i) and intermediate dose group (j-l). (m-o) Histological analysis using Prussian blue staining for iron particles shows cytosolic deposition of blue crystals in the grafted stem cells and migration toward the infarcted brain (asterisk in m, n). The interrupted line in (o) shows the boundary of infarcted brain. Bars= (n) 50 μm; (o) 20 μm. Reprinted with permission from M. M. Daadi, Molecular and magnetic resonance imaging of human embryonic stem cell-derived neural stem cell grafts in ischemic rat brain, Mol Ther 17 (2009) 1282–91 (41).

Figure 4

Bioluminescence and fluorescence imaging of transplanted stem cells. (a) Ex vivo analysis shows a linear correlation between cell number and bioluminescence imaging signal. The total count is noted above the corresponding well, with the color scale bar denoting the range of signal in photons per second per cm² per steradian. (b) Fluorescent microscopy shows bright, cytosolic enhanced green fluorescent protein expression, with corresponding nuclei stained with blue 4, 6-diamidino-2-phenlindole. Scale bar: 5 μm. (c) FACS analysis demonstrates robust expression of enhanced green fluorescent protein by more than 87% of cells from FVB wild type mice and L2G transgenic mice. (d) Bioluminescence imaging shows that transplanted bone marrow mononuclear cells accurately home to areas of injury. Images of the sham (left) and ischemic reperfusion injury (right) are shown following stem cell transplantation (day 1 to 28). There is a persistent elevation of signal through day 14, which gradually decreases by day 28. (Please note the maximum values for scale bars in p/s/cm²/sr are different in the three rows). Abbreviations: p/s/cm²/sr, photons per second per cm² per steradian. Reprinted with permission from Sheikh, A, et al. Molecular imaging of bone marrow mononuclear cell homing and engraftment in ischemic myocardium, Stem Cells 25 (2007) 2677–84 (56).

Figure 5

Tracking of survival and engraftment of transplanted islets by PET reporter labeling. (a) Immunohistological analysis of engineered HSV1-tk expressing islets, performed 15 days after transplantation, shows expression of thymidine kinase (shown in green) and insulin (red), observed in scattered islets. (b) Longitudinal micro PET imaging of a mouse with an intrahepatic islet graft shows that signals from the liver area of HSV1-tk expressing islets gradually decline over time. When imaged 40 days after transplantation, signals from the liver area were essentially at background levels. Signal loss was likely due to cellular death shortly after implantation and the transient nature of viral gene expression. Reprinted with permission from Y. Lu, et al., Noninvasive imaging of islet grafts using positron-emission tomography, Proc Natl Acad Sci U S A 103 (2006) 11294–9 (58).