Assessment of human multi-potent hematopoietic stem/progenitor cell potential using a single in vitro screening system

Abstract

Hematopoietic stem cells are responsible for the generation of the entire blood system through life. This characteristic relies on their ability to self renew and on their multi-potentiality. Thus quantification of the number of hematopoietic stem cells in a given cell population requires to show both properties in the studied cell populations. Although xenografts models that support human hematopoietic stem cells have been described, such in vivo experimental systems remain restrictive for high throughput screening purposes for example. In this work we developed a conditional tetracycline inducible system controlling the expression of the human NOTCH ligand Delta-like 1 in the murine stromal MS5 cells. We cultured hematopoietic immature cells enriched in progenitor/stem cells in contact with MS5 cells that conditionally express Delta-like 1, in conditions designed to generate multipotential lineage differentiation. We show that upon induction or repression of DL1 expression during co-culture, human immature CD34(+)CD38(-/low)(CD45RA(-)CD90(+)) cells can express their B, T, NK, granulo/monocytic and erythroid potentials in a single well, and at the single cell level. We also document the interference of low NOTCH activation with human B and myelo/erythroid lymphoid differentiation. This system represents a novel tool to precisely quantify human hematopoietic immature cells with both lymphoid and myeloid potentials.

Figure 1. Characterization of MS5/DL1^ind cells lines.

(A) Schematic representation of the TET/on lentiviral DL1 vector system. (B–D) Measure of DL1 expression in established MS5 cells. (B) Cell lines, previously transduced with 100 (/DL1^ind100), 500 (/DL1^ind500) or 1000 (/DL1^ind1000) ng P24 vectors, were cultured in presence (+) or absence (−) of 1 µg/ml of doxycyclin during 48 hours. Proteins were extracted and DL1 expression was analysed by western blot. (C) Follow up of DL1 induction. MS5/DL1^ind100–500 cells cultured with (+) or without (−) doxycyclin (1 µg/ml) were lysed at different times (in hours) after adding doxycyclin to the culture medium. (D) Follow up of DL1 drop down expression according to time after induction. DL1 expression was induced 24 hours before washing out medium (time 0 hour). At different time points, cells were harvested and protein extracted to follow up decrease of DL1 expression. (E) Surface expression of human DL1 in MS5 cell lines detected by flow cytometry. Shown are histograms of DL1 expression levels in presence of 1 µg/mL doxycycline (+ condition), after 48 hours of induction (- ->+condition) and 72 hours after washing out doxycycline from the medium (+ -> - condition). Arrows indicate positive and negative DL1 expression on cells. (F) Follow up on DL1 surface expression as a function of time after adding (+ doxycyclin) or washing out (-doxycyclin) doxycycline in the culture. Shown are % of DL1⁺ cells measured as in (E).

Figure 2. T cell differentiation from CD34⁺CD38^−/low cells cultured with MS5 cell lines in presence of 1 µg/ml doxycyclin.

(A) 5000 cells were cultured in complete medium with MS5/DL1 and MS5/DL1^ind cell lines. Flow cytometry was performed on the progeny of such cells 21 and 42 days after initiating the cultures. Shown are results of labelled cells with anti-human specific antibodies directed against CD34, CD7, CD4, CD8 and CD3. % of positive cells are indicated beside each quadrant. (B) Summary results of analysing the progeny of 5–10×10³ CD34⁺CD38^−/low cells from 7–8 individual CB samples. Left: shown are % of CD7⁺ cells and every symbols represent an individual sample. Right, Proportion of indicated populations gated in the CD7⁺ cells for every time point tested. Shown are mean +/− SEM (C) Levels of NOTCH target gene transcripts at 21 days of culture with doxycyclin. Results were normalized over ß2m transcripts levels for each sample. (D) Follow up of T cell differentiation after switching DL1 expression off (MS5 or MS5/DL1^ind – doxycyclin) during culture. Shown are results from 1 out of 2 experiments. Flow cytometry was performed at 3 time points for the measure of CD4 and CD8 expression on human cells. % of positive cells are indicated beside every dot plot. When a specific gate was drawn, % of cells is indicated beside the gate. K&W statistical analysis was used for data of this figure.

Figure 3. Human B, myeloid and erythroid development from CD34⁺CD38^−/low(CD45RA⁺CD90^+/−) cells cultured with MS5 and MS5/DL1^ind cells.

5×10³ CD34⁺CD38^−/low(CD45RA⁺CD90^+/−) cells were cultured in absence of DL1 expression (MS5 or – doxycyclin) during 21 days. Cells were harvested and analysed for the expression of CD19 (B cells), CD14 and CD15 (Myeloid cells) and CD36/GPA (erythroid cells). (A) Shown are results from a representative experiment performed with CD34⁺CD38^−/low cells. (B) Summary of results of cultures of 1–5×10³ CD34⁺CD38^−/low cells from 3–5 CB samples. (C) Results from cultures of 1–5×10³ CD34⁺CD38^−/lowCD45RA⁻CD90⁺ or CD90⁻ cells. Shown of mean % of cells obtained with 3 CB samples. (D) Transcripts levels in CD34⁺CD38^−/low cells cultured 21 days with MS5 cell lines in absence of doxycyclin. Results are normalized over ß2m expression levels. (E) Characterization of B cell progenitor populations generated during cultures of 10×10³ CD34⁺CD38^−/low cells and harvested at 21 days. Shown are means of 2 experiments performed with distinct CB samples. M&W and K&W statistics were respectively used for data in (C) and (B, D, E).

Figure 4. Multi-lineage differentiation of human CD34⁺CD38^−/low cells in co-cultures with MS5 cell lines.

Sorted cells (10×10³ cells/well) were cultured 21 days without DL1/expressing stromal cells (MS5 or MS5/DL1^ind100–doxycyclin). Cells were harvested and analysed by FACS for the presence of myeloid (CD14⁺/CD15⁺), lymphoid B (CD19⁺) and progenitor (CD34⁺) cells. Half of cells were plated with DL1/expressing stromal cells (MS5/DL1 or MS5/DL1^ind100+ doxycyclin). (A) Results of a representative experiment. % of expressing cells are indicated under the plots in case of quadrant stat or beside the gated population. (B) Summary results of 3 CB samples. (C) Levels of Gata3, pTα, TCF7 and Hes1 transcripts in the progeny of CD34⁺CD38^−/low cells before (MS5 or MS5/DL1^ind100–doxycyclin) and after (MS5/DL1 or MS5/DL1^ind100+doxycyclin) DL1 induction during culture. Results were normalized over ß2m transcript levels. M&W and K&W statistical analyses were respectively used in (B) and (C).

Figure 5. Single cell cultures of CD34⁺CD38^−/low cells with MS5/DL1^ind100 cells.

(A) Experimental design and analysis of 3 representative clones by FACS before (step 1: MS5/DL1^ind100–doxycyclin, day 21) and after (step 2: MS5/DL1^ind100+doxycyclin, day 42) induction of DL1 during culture. G/M, CD14⁺/CD15⁺; B, CD19⁺; T, CD5⁺CD4⁺CD8^+/; NK, CD56⁺CD5⁻. (B) Distribution of different clones obtained in the culture of 180 CD34⁺CD38^−/low cells, among which 57 proliferated enough to allow FACS analysis at 21 days. Indicated is the proportion of different potentials measured in the proliferating clones.