2.1.

Animals

Mice used in this study were C57BL/6J and B.6SJL/Boy strains purchased from Jackson Laboratory (Bar Harbor, Maine). All experimental animals were housed in a conventional clean facility using barrier mouse housing facilities [Tecniplast (Exton, Pennsylvania) individually ventilated chambers], and given ad-libitum access to autoclaved mouse chow and acidified water.

2.2.

Isolation of $\mathrm{Fr}25{\mathrm{lin}}^{-}$ Cells

Whole bone marrow cells were harvested by flushing medullar cavities of femurs, tibias, and iliac bones. Single cell suspensions of $5\text{to}10\times 10^8$ wBMC from ten mice were loaded into the chamber of a counterflow centrifuge (Beckman Instruments, Palo Alto, California) at a flow rate of $15\mathrm{ml}∕\min$ with a constant rotor speed of $3000\mathrm{rpm}$ at RT. The smallest subset of nucleated cells (Fr25) were collected at a rate of $25\mathrm{ml}∕\min$ in $200\mathrm{ml}$ . Lineage-positive cells, including T and B lymphocytes, macrophages, granulocytes, and erythroid cells, were immunomagnetically depleted. The Fr25 cells were incubated for $30\min$ at $4°\mathrm{C}$ with rat antimouse monoclonal antibodies (mAb) against GR-1 (RB6-8C5), Mac-1 (M1/70), B220 (RA3-3A1/6.1), and CD-5 (53-7.3) extracted from hybridoma cell lines (ATCC, Rockville, Maryland), and purified Ter-119 (eBioscience Incorporated, San Diego, California). Antibody-coated cells were incubated with sheep antirat magnetic beads ( $4\times {10}^7∕{10}^7$ cells) (Dynal Biotech ASA, Oslo, Norway) at $4°\mathrm{C}$ for $15\min$ . Rosetted cells were precipitated by exposure to a magnetic field, and the supernatant containing lineage-negative cells was collected. The isolated cells were evaluated for viability by the trypan blue exclusion test and evaluated for size characteristics and expression of hematopoietic lineage markers using a Vantage SE flow cytometer (Becton Dickinson, Franklin Lakes, New Jersey).

2.3.

Fourier Transform Infrared Microscopy Measurements and Data Analysis

$1.5\mu \mathrm{l}$ of packed cells were deposited on a zinc selenide (ZnSe) slide to form an approximate monolayer of cells, and then air dried for $15\min$ under laminar flow to remove water. Measurements of selected microscopic sites with an aperture of $100\mu \mathrm{m}$ and similar ADC rates were performed using the FTIR microscope IR scope 2 (Bruker Optik GmbH, Ettlingen, Germany) in transmission mode as described previously.²⁰ FTIR microscopy (MSP) was chosen as a favorable methodology for this study, since other systems such as FTIR-attended total reflectance (ATR) which can measure cells in their native culture media, are limited in their sensitivity due to water interference, restricted penetration depth of the evanescent waves into the sample (about $3\text{to}4\mu \mathrm{m}$ ), and higher contribution from scattering effects compared to FTIR-MSP.²¹

The spectra were baseline corrected using a polynomial rubber band with 64 points (OPUS software Bruker Optik GmbH, Ettlingen, Germany) and were vector normalized in the region $700\text{to}4000{\mathrm{cm}}^{-1}$ or min-max normalized in the region of $700\text{to}1800{\mathrm{cm}}^{-1}$ . To obtain precise absorbance values at a given wave number with minimal background interference, the second derivative spectra were used to determine intensities of the biomolecules of interest. The second derivative method is highly susceptible to changes in full width at half maximum (FWHM) of the IR bands. However, in the case of biological samples, all cells from the same type are composed of similar basic components, which give relatively broad bands. Thus, there is a possibility of neglecting the changes in the band FWHM.²²

2.4.

Statistics

Data are presented as the $\text{mean}\pm \text{standard}$ error (SEM) derived from at least three independent experiments. Statistical analysis was performed using the student t-test. $P$ -values $<0.05$ were considered significant. Linear regressions were done by the least-squares method, and the data analysis package (Origin, Micro-Cal, Incorporated, Northampton, Massachusetts) was used when comparing a series of datasets. Data reduction by principal component analysis (PCA) was implemented as previously described.²³

3.1.

Characterization of Hematopoietic Stem Cells by Flow Cytometry

To obtain fractions of bone marrow cells enriched in primitive stem cells, we adopted a technique of counterflow centrifugal elutriation.²⁴ The elutriated fraction collected at a flow rate of $25\mathrm{ml}∕\min$ was immunomagnetically depleted of cells expressing hematopoietic lineage markers using monoclonal antibodies against granulocytes, macrophages, T and B lymphocytes, and erythroid cell-specific antigens. The size characteristics of wBMC and $\mathrm{Fr}25{\mathrm{lin}}^{-}$ cells as determined by flow cytometric analysis of side and forward scatter parameters are demonstrated in Fig. 1 . $\mathrm{Fr}25{\mathrm{lin}}^{-}$ cells represented the smallest fraction of the unfractionated wBMC population. Bone marrow is a heterogeneous population containing both hematopoietic and nonhematopoietic cells. To evaluate the hematopoietic contents of the $\mathrm{Fr}25{\mathrm{lin}}^{-}$ subset, expression of the pan-hematopoietic marker CD45 was examined and compared to whole BMC. Both BMC and $\mathrm{Fr}25{\mathrm{lin}}^{-}$ cells were composed of a major hematopoietic population (96 and 81%, respectively).

Fig. 1

Evaluation of cell size and expression of hematopoietic markers in wBMC and Fr25lin^- cells. Whole bone marrow (wBMC) and elutriated lineage-depleted Fr25 (Fr25lin^-) cells were stained with a cocktail of monoclonal antibodies against hematopoietic lineage markers (GR-1, Mac-1, B220, Ter-119, and CD5) and CD45.

3.2.

Fourier Transform Infrared Microscopy Analysis of Hematopoietic Stem Cells

Biochemical analysis of extracted HSCs and comparison to wBMC was conducted using FTIR-MSP. FTIR-MSP spectra of wBMC and HSCs in the regions $3000\text{to}2800{\mathrm{cm}}^{-1}$ and $1800\text{to}700{\mathrm{cm}}^{-1}$ after baseline correction and min-max normalization to amide II are shown in Fig. 2 . The spectra exhibits significant differences between wBMC cells and HSCs at several wave numbers; each corresponds to specific functional groups related to fundamental cellular components such as lipids, proteins, and carbohydrates/nucleic acids.

Fig. 2

Fourier transform infrared (FTIR) spectra of bone marrow (BM) cells and hematopoietic stem cells (HSCs). (a) Representative FTIR spectra exhibit significant differences between BM cells and HSCs after baseline correction and amide-II normalization. Each spectrum represents the average of five measurements at different sites for each sample. The main absorbance bands are marked. (b) Second derivative extended spectra of BM cells and HSCs after baseline correction and vector normalization.

The region $3000\text{to}2800{\mathrm{cm}}^{-1}$ contains absorbance bands due to symmetric and asymmetric $\mathrm{C}{\mathrm{H}}_3$ and $\mathrm{C}{\mathrm{H}}_2$ stretching vibrations related mainly to proteins and lipids, respectively.²⁵ The absorbance ratio of methylene to methyl bands quantified by the bands integrated absorbance at 2960 and $2873{\mathrm{cm}}^{-1}$ divided by 2922 and $2852{\mathrm{cm}}^{-1}$ was higher for the HSCs than for BM $(p<0.05)$ . Although the parameter of lipids/proteins is significant for characterizing HSCs, the difference between HSCs and BM cells is quite small, thus, there is a need for a better biochemical parameter.

The phosphate bond absorption at $\sim 1237{\mathrm{cm}}^{-1}$ (asymmetric vibration) and $1088{\mathrm{cm}}^{-1}$ (symmetric vibration) is more prominent in the HSCs spectra than in the wBMC spectra, as presented in Figs. 2 and 2. These bands are related mainly to nucleic acids and carbohydrates.^{25, 26} Another band that is more prominent in HSCs is located at $966{\mathrm{cm}}^{-1}$ and corresponds to C–C/C–O stretching of deoxyribose-ribose skeletal vibration of the DNA.²⁷

3.3.

Principal Component Analysis and Statistical Analysis of Fourier Transform Infrared Microscopy Spectra

To determine the main spectral characteristics of HSCs and BM cells, unbiased PCA analysis was conducted. The FTIR-MSP spectra are composed of 286 vector components corresponding to absorbance intensities. PCA analysis reduces the number of variables to six principle components (PCs).²⁴ The best separation between HSC and BM cell spectra was achieved by PC4 versus PC1, as presented in Fig. 3 . PC1 adequately separates between the two groups of cells, while PC4 provides the heterogeneity of each one of the individual groups. According to PC4, HSCs have less heterogeneity than the total BM cell population. The loading of each PC reveals the main vibrational bands for distinction between HSCs and BM cell spectra [Fig. 3]. As observed in Fig. 3, there are several major bands contained in the PC1 loadings such as 966, 1088, and $1240{\mathrm{cm}}^{-1}$ , which correspond to nucleic acids/carbohydrates, and the $\sim 1678{\mathrm{cm}}^{-1}$ band, which corresponds to untiparallel $\beta$ -sheets of proteins and to $\mathrm{C}\mathrm{O}$ stretching vibrations that are hydrogen bonded.²⁵ PC4 loadings show a different set of bands [Fig. 3] as expected. The later contributes mainly to the heterogeneity of both HSCs and BM cells.

Fig. 3

Statistical analysis of FTIR-MSP spectra. (a) PCA was employed on FTIR-MSP spectra composed of 286 vector components, reducing it to six PCs, which describe 98.1% of the data variance. PC4 versus PC1 was found to give the best separation between HSCs and BM cell spectra. (b) Absolute loading values of PC1 and PC4 in the region $700\text{to}1800{\mathrm{cm}}^{-1}$ . (c) The absorbance bands at 781, 966, and $1085\text{-}{\mathrm{cm}}^{-1}$ , which are mainly related to DNA, are more prominent in the HSCs spectra than in the BM spectra. Values plotted are the means ±SEM derived from at least three independent experiments. $({}^{*}P<0.001,{}^{**}P<0.05)$ .

The bands that give the finest distinction according to the PCA analysis correspond mostly to the DNA. A student t-test was employed to determine the ability of these bands to serve as markers for identifying HSCs in the BM using FTIR-MSP, as presented in Fig. 3. To verify that these biomarkers correspond to DNA, an additional characteristic band of DNA at $780{\mathrm{cm}}^{-1}$ was also tested and was found to be appropriate for characterizing HSCs [Figure 3].²⁷

3.4.

Quantification of Hematopoietic Stem Cells by Fourier Transform Infrared Microscopy

To evaluate the utility of FTIR for quantifying the number of HSCs from wBMC (which also contain $0.3\pm 0.1\mathrm{\%}$ of HSCs)¹¹ based on the characteristic biomarkers of HSCs, we aimed to determine the minimal detectable percentage of HSCs in the BM. For this goal, HSCs were diluted with BM in decreasing percentages and measured by FTIR. Several wave numbers (1088, 966, and $780{\mathrm{cm}}^{-1}$ ), which were found to be the most significant markers to characterize HSCs, were used in determining the maximal sensitivity of FTIR for HSCs quantification.

Figure 4 shows the characteristic spectra obtained with different concentrations of HSCs diluted in BM cells, and a correlation curve between HSC percentages in the BM and the respective absorbance at $966{\mathrm{cm}}^{-1}$ [Fig. 4]. To evaluate the utility of quantifying HSCs in the BM, we calculated a linear regression curve for each single experiment, excluding errors due to the complicated cell extraction procedure. A good correlation was achieved $(R^2>0.98)$ . Using this spectral parameter, we were able to differentiate $(p<0.05)$ between samples diluted up to approximately 0.1% HSCs compared with pure BM samples. However, due to the complexity of this experiment, the exact sensitivity and accuracy limits of FTIR-MSP in detection of HSCs in BM should be further investigated.

Fig. 4

Quantification of HSCs in whole BM cell populations by FTIR-MSP. Isolated HSCs were diluted (from 0 to 100% HSCs) in whole BM cell population. (a) Second derivative extended representative spectra of HSCs and BM cells after baseline correction and vector normalization. (b) A linear correlation between the absorbance band of the DNA at $967{\mathrm{cm}}^{-1}$ and the percentage of HSCs in BM cell population. Values plotted are the means ±SEM derived from at least three independent experiments.