Assay for identifying colony-forming cells

Abstract

The present invention relates to a method for detecting differentiated hematopoietic cells, comprising the following steps: a) separating undifferentiated hematopoietic stem cells in groups of 1 - 1000 cells on a support, b) proliferating the separated cells by providing a cell culture medium containing growth factors to form cell colonies of differentiated hematopoietic cells, c) contacting the cell colonies with one or more labeled conjugates, the labeled conjugates comprising at least one detection moiety and at least one antigen recognition moiety directed against CD14, CD235a and CD15, d) detecting the relative amount of differentiated hematopoietic stem cells in the cell colonies labeled with the labeled conjugates.

Description

Technical Field

[0001] The present invention relates to assays and methods for detecting or identifying colony-forming cells and differentiating colonies derived from those cells, particularly hematopoietic stem cells (HSCs). Background Art

[0002] Hematopoietic stem cells (HSCs), like every stem cell, can self-renew and at the same time give rise to hematopoietic progenitor cells, which will subsequently and ultimately differentiate into the lymphoid lineage (giving rise to T-, B-, and NK-cells), and into the myeloid lineage (giving rise to red blood cells, platelets, granulocytes, macrophages, etc.). After long-term experiments, it became clear that in humans, the cells with the ability to differentiate into several multi-lineage progenitor cells are the CD34+ cell population. In fact, this has been confirmed both in vivo in mouse models and in the context of hematopoietic stem cell transplantation (HSCT). The most reliable, easy, and long-existing assay in vitro is the colony-forming cell (CFC) assay, also known as the methylcellulose assay. This assay is based on the ability of CD34+ cells to differentiate into different colonies, which can then be counted and characterized.

[0003] In the classical CFC assay, a certain number of CD34+ cells (usually between 250 - 500 cells) are plated in 1.1 ml of a medium capable of supporting myeloid lineage differentiation in a 35 mm dish. This medium consists of IMDM supplemented with cytokines (such as SCF, Flt3 ligand, TPO, IL3, and IL6, etc.), fetal bovine serum, and methylcellulose to prepare a medium with high viscosity. The idea behind this is that the cells will not be able to move freely within the liquid medium, but rather the cells will remain in a certain position within the methylcellulose semi-solid medium. Thus, after incubation for about 14 days, the 35 mm dish will contain many different colonies, at least theoretically each colony derived from a single CD34+ cell, making it possible to subsequently measure the percentage of CD34+ cells capable of forming colonies and the colony types. Thus, after observation using an inverted microscope, based on morphological and phenotypic criteria, and on the number and type of mature cells they contain, the colonies derived from different types of progenitor cells are classified and counted. Figure 1 Shows typical results of a CFC colony assay from a 35 mm dish. The dots represent different types of colonies.

[0004] The colonies formed in the known methylcellulose CFU assay are classified into the following categories: colony-forming unit-erythroid (CFU-E), burst-forming unit-erythroid (BFU-E), colony-forming unit-macrophage (CFU-M), granulocyte-macrophage colony-forming unit (CFU-GM), and granulocyte-erythroid-macrophage-megakaryocyte colony-forming unit (CFU-GEMM).

[0005] BFU-E contains more than 200 early erythroblasts and is usually present in 3 - 8 densely packed clusters. CFU-E is smaller than BFU-E and contains 8 - 200 erythroid progenitor cells, usually present in one or two densely packed clusters. Due to the hemoglobin-containing cells, both CFU-E and BFU-E have a dark red / orange to brown color. CFU-GEMM presents a dense area, which is usually located at the center of the peripheral flat lawn of translucent cells, and the translucent cells can be large or small ("fried egg" appearance).

[0006] Because CFU-GEMM also contains erythroblasts (depending on the level of hemoglobinization), the color of the hemoglobin-containing cells can vary from dark red / orange to brown. CFU-G is usually flat and consists of 20 - 40 small translucent cells with a germinative center. Finally, CFU-M is a sparsely growing flat colony consisting of >20 large translucent cells, while CFU-GM is also a flat colony consisting of 20 - 50 small and large translucent cells. Figure 1 Describes typical results of CFU assays, where different types of colonies can be seen and ultimately counted only based on the morphological and phenotypic criteria as described above. Figure 2 Shows different colony types at a higher magnification.

[0007] Unfortunately, the current way of counting colonies by using morphological and phenotypic criteria can be biased and largely depends on the experience of the person counting them. To determine the degree of variability, we conducted several experiments in which we asked several researchers to count the same 35 mm dishes containing colonies after a 14-day incubation period.

[0008] As shown in Figures 3(a and b) from a representative experiment, after counting and characterizing the same colonies, the results showed a very high degree of variability in identifying the total number of colonies and assigning colonies to the correct type. More specifically, only 6 out of a total of 27 colonies were correctly counted and evaluated, and less than a quarter of the colonies had a clear identification. Clearly, BFU-E type colonies are the easiest colonies to identify, producing accurate and reproducible results. These findings strongly suggest that morphological and phenotypic classification is neither robust nor reproducible. Summary of the Invention

[0009] Accordingly, an object of the present invention is a method for detecting differentiated hematopoietic cells, comprising the following steps:

[0010] a) Separating undifferentiated hematopoietic stem cells in groups of 1 - 1000 cells on a support,

[0011] b) proliferating the isolated cells by providing a cell culture medium containing growth factors to form a cell colony of differentiated hematopoietic cells,

[0012] c) contacting the cell colony with one or more labeled conjugates, the labeled conjugates comprising at least one detection moiety and at least one antigen recognition moiety directed against CD14, CD235a, and CD15,

[0013] d) detecting the relative amount of differentiated hematopoietic stem cells in the cell colony labeled with the labeled conjugate.

[0014] Another object of the present invention is the use of a method for determining the differentiation state of stem cells in a cell sample.

[0015] Another object of the present invention is a labeling mixture comprising one or more labeled conjugates, each of the labeled conjugates comprising at least one detection moiety and at least one antigen recognition moiety directed against CD14, CD235a, and CD15.

[0016] Another object is a kit for detecting differentiated hematopoietic stem cells, which comprises a cell culture medium having at least one growth factor and a labeling mixture, the labeling mixture comprising one or more labeled conjugates, each of the conjugates comprising at least one detection moiety and at least one antigen recognition moiety directed against CD14, CD235a, and CD15.

[0017] In the method of the present invention, a cell culture medium containing growth factors is used. Such cell culture media are known to those skilled in the art, and exemplary compositions are shown in the Examples. Hereinafter, the cell culture medium containing growth factors is referred to as "HSC-CFU assay medium" or "assay medium". Such a medium is available from Miltenyi Biotec B.V. & Co. KG under the trade name "StemMACS HSC-CFU assay medium".

[0018] The labeling mixture of the present invention or for use in the present invention is referred to as "antibody mixture" or "HSC-CFU antibody mixture", and is available from Miltenyi Biotec B.V. & Co. KG under the trade name "StemMACS HSC-CFU antibody mixture". BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shows typical results of a CFC colony assay from a 35 mm dish. The dots represent different types of colonies.

[0020] Figure 2 : Different types of colonies from a CFU assay at a higher magnification and their specific characteristics.

[0021] Figure 3: Results obtained after six independent researchers evaluated the same 35 mm dishes. The colonies were first counted and then listed in order so that each researcher could characterize each colony according to type. Figure 3a The dish with the counted colonies is shown. Figure 3b Results obtained from 6 independent researchers are shown, demonstrating the high variability observed after visual inspection. These findings strongly suggest that morphological and phenotypic classification is neither robust nor reproducible.

[0022] Figure 4 : When inoculating the same cell concentration, the average total colony counts found in two 35 cm wells were compared with the average total colony counts found in three 96-well plates. Error bars represent standard deviation.

[0023] Figure 5 : Gating strategy. The regions required for analysis are highlighted.

[0024] Figure 6 : BFU-E characterization. BFU-E colonies are positive for the erythroid marker CD235a.

[0025] Figure 7 CFU-G characterization is shown.

[0026] Figure 8 CFU-M characterization is shown. CFU-M colonies should be at least 50% positive for CD14.

[0027] Figure 9 CFU-GM characterization is shown. It can be characterized as CFU-GM when the colony expresses more than 30% CD14-positive cells and more than 30% CD15-positive cells. It should also be negative for the erythroid marker CD235a. Detailed Description

[0028] To address the issue of low reproducibility in colony evaluation and to increase robustness and eliminate bias in CFU assays, we have developed a method according to the present invention. The method according to the present invention provides a highly standardized method for analyzing hematopoietic stem and progenitor cells, as it combines differentiation in cell culture with a standardized, flow cytometry-based readout and eliminates the need for user-dependent visual scoring under the microscope.

[0029] The method of the present invention allows for the detection or identification of colony-forming cells and optionally the differentiation of colonies derived from those cells, particularly hematopoietic stem cells (HSCs).

[0030] Undifferentiated or differentiated hematopoietic cells are preferably undifferentiated or differentiated hematopoietic stem cells.

[0031] In step a) of the method of the present invention, the cells are separated on the surface in groups of 1 - 1000 cells. Preferably, the number of cells is smaller, such as 100 to 500 cells or 150 to 350 cells.

[0032] For colony formation, the cells can be diluted in HSC - CFU assay medium without methylcellulose and deposited at a concentration of 2.5 cells / well in, for example, a 96 - well plate. In this way, each well corresponds to the clonal progeny of a single hematopoietic stem cell or progenitor cell. During the subsequent incubation period (14 days), the HSC - CFU assay medium promotes the growth and differentiation of the cells deposited in suspension. After they are formed, the colonies are further evaluated according to their type by staining each well of the 96 - well plate with an HSC - CFU antibody mixture and then analyzed by flow cytometry.

[0033] Thus, each colony can be easily identified by the corresponding marker combination. In addition, based on the frequency of each colony type, the percentage of occurrence of each colony can be determined relative to the total number of colonies. This setup provides a standardized, user - independent analysis of the HSC - CFU assay and allows automation in combination with any flow cytometer.

[0034] Using the method of the present invention, it is possible to determine the differentiation status of stem cells in a cell sample. For this purpose, the differentiated hematopoietic stem cells in the cell colonies are detected as CFU - GEMM, CFU - GM, CFU - M, BFU - E, and CFU - G by the relative amounts of cells labeled with marker conjugates as defined in the following table.

[0035]

[0036] Table 3 Colony detection parameters. The differences from 100% relate to other antigens not of interest in the method of the present invention.

[0037] Preferably, the method according to the present invention is carried out in the absence of methylcellulose.

[0038] Depending on the source of the cell sample, it is recommended to remove red blood cells and / or enrich CD34+ cells before applying the method to the cell sample.

[0039] The removal of red blood cells can be carried out by lysing or precipitating the red blood cells from the sample. These techniques are known to those skilled in the art.

[0040] Enrichment of CD34+ cells from a cell sample containing undifferentiated hematopoietic stem cells is preferably carried out to at least 50% purity, more preferably to at least 90% purity. Enrichment methods, such as by magnetic cell sorting or flow cytometer known to those skilled in the art.

[0041] The detection moiety of the labeled conjugate has no particular relevance to the method and may optionally be a chromophore moiety, a fluorescent moiety, a phosphorescent moiety, a luminescent moiety, an absorptive moiety, a radioactive moiety, a transition metal, and an isotope mass tag moiety. However, a labeled conjugate comprising one or more fluorescent moieties is preferred.

[0042] This also applies to the antigen recognition moiety of the labeled conjugate, which may be selected from antibodies, fragmented antibodies, fragmented antibody derivatives, peptide / MHC complexes targeting TCR molecules, cell adhesion receptor molecules, receptors for co-stimulatory molecules, or artificially engineered binding molecules.

[0043] Regarding the labeled mixtures and kits of the present invention, it is preferred to provide at least 3 different labeled conjugates, each conjugate comprising at least one antigen recognition moiety directed against CD14, CD235a, and CD15, respectively. A person skilled in the art can determine the relative amounts of the labeled conjugates to obtain optimal results. The ratio between the labeled conjugates directed against CD14, CD235a, and CD15 is preferably 55 - 75%, 15 - 30%, and 5 - 20% by weight.

[0044] Examples

[0045] Materials and Methods

[0046] The HSC-CFU assay medium is a culture medium preparation that supports the growth of human BFU-E, CFU-E, CFU-G, CFU-M, CFU-GM, and CFU-GEMM colonies. A typical composition is shown in Table 1.

[0047] Components Concentration in the medium Fetal bovine serum (FBS) 30％ Bovine serum albumin (BSA) 1％ L-Glutamine 2 mM 2-Mercaptoethanol 0.1 mM Stem cell factor (SCF) 50 ng / ml GM-CSF 20 ng / ml G-CSF 20 ng / ml IL-3 20 ng / ml IL-6 20 ng / ml Erythropoietin (Epo) 3 U / ml

[0048] Table 1

[0049] The following reagents and instruments are required, some of which are optional

[0050] · A flow cytometer capable of differentiating APC, PE, and VioBlue, such as the MACSQuant Analyzer

[0051] · When using the MACSQuant Analyzer 10, an accessory for handling 96-well plates on the flow cytometer, such as Chill 96 Rack (#130 - 094 - 459)

[0052] · Sterile 15 polypropylene tubes

[0053] · Sterile disposable pipette tips

[0054] · Sterile pipettes

[0055] · 96-well round bottom plates

[0056] · Humidity chamber

[0057] · Dilution medium: Iscoves Modified Dulbecco's Medium (IMDM)

[0058] · PBS / EDTA buffer containing 0.5% BSA (PEB), sterile and non-sterile

[0059] · Sterile water

[0060] · Multichannel pipette

[0061] · Reagent reservoir

[0062] · CD34-APC

[0063] · CD45-FITC for optional white blood cell measurement. Some CD45-positive cells are also CD34-positive

[0064] · FCR blocker

[0065] · (Optional) Red blood cell lysis buffer (10x) (130 - 094183)

[0066] Preparation of HSC-CFU assay medium

[0067] To avoid repeated freeze-thaw cycles, the HSC-CFU assay medium should be aliquoted into appropriate volumes. The protocol is as follows: 1. Thaw the medium overnight at 4°C.

[0068] 2. Vigorously shake the bottle.

[0069] 3. Using a sterile pipette, aliquot it into sterile tubes (15.0 ml / tube).

[0070] 4. Freeze the aliquots at -20°C. Thaw at room temperature or overnight at 4°C before use.

[0071] Preparation of cell samples

[0072] Hematopoietic colony formation assays can be performed using monocytes from bone marrow, cord blood, or peripheral blood. Similarly, enriched hematopoietic stem and progenitor cells, such as enriched lineage marker-negative (lin–), CD133+ or CD34+ cells, or ES and iPS cell-derived progenitor cells, can be used.

[0073] For pre-enrichment of CD133+, CD34+ or lin– cells, refer to the data sheet of the corresponding isolation product.

[0074] Settings for HSC-CFU assay

[0075] The protocol is as follows

[0076] 1. Thaw the required number of aliquots of HSC-CFU assay medium at room temperature or overnight at 4°C. Each 15 mL aliquot corresponds to 1 test / assay and is sufficient to process three 96-well plates. (Optional) For samples with high red blood cell content, it is highly recommended to perform red blood cell lysis prior to determining the CD34+ cell count. Follow the instructions for the red blood cell lysis buffer (10x). Resuspend the cell pellet in sterile PEB.

[0077] 2. Under sterile conditions, take up to 20 μl of the small sample and determine the cell number.

[0078] 3. Using sterile technique, take an aliquot of up to 10 6 cells and proceed to steps 4-5.

[0079] 4. Centrifuge at 300 x g for 10 minutes. Aspirate the supernatant completely.

[0080] 5. Resuspend in 96 μl of non-sterile PEB.

[0081] 6. Add 2 μl of CD34-APC and 2 μl of CD45-FITC.

[0082] 7. Mix well and incubate in the refrigerator (2-8°C) in the dark for 10 minutes.

[0083] 8. Wash the cells by adding 1-2 mL of buffer and centrifuge at 300 x g for 10 minutes. Aspirate the supernatant completely.

[0084] 9. Resuspend the cell pellet in an appropriate amount of non-sterile PEB for flow cytometry analysis. Adjust the cell concentration to 250 CD34+CD45+ cells per 1 mL of medium using normal IMDM.

[0085] 10. Note: For each sample, three 96-well round bottom plates are required. This corresponds to 1000 CD34+ cells in 4 ml of IMDM.

[0086] 11. Vortex the tube to ensure even distribution of the cells.

[0087] 12. Transfer the cell suspension to the reagent reservoir.

[0088] 13. Pipette 10 μl into each well of three 96-well round bottom plates

[0089] 14. Transfer 15 mL of HSC-CFU assay medium to a new reagent reservoir.

[0090] 15. Pipette 50 μl into each well of three 96-well round bottom plates

[0091] 16. Place the plate in a humidity chamber or sterile hood containing several milliliters of sterile water to minimize evaporation of the cell culture medium during incubation.

[0092] 17. Incubate the plate in a humidified incubator at 37 °C and 5% CO2 for 12 - 14 days.

[0093] Flow cytometry analysis

[0094] The protocol is as follows

[0095] 1. Prepare the fluorescent dye mixture by diluting 45 μl of the HSC - CFU antibody mixture to a final volume of 4.5 mL with PBS / EDTA / 0.5% BSA buffer.

[0096] 2. Add 15 μl of the diluted mixture to each well of three 96 - well round - bottom plates.

[0097] 3. Add 25 μl of PBS / EDTA / 0.5% BSA buffer to each well to a total volume of 100 μl.

[0098] 4. Continue the acquisition.

[0099] 5. Note: Ensure that the flow cytometer has been set up for processing 96 - well plates. If supported by the flow cytometer, a gentle mixing mode is recommended.

[0100] Settings for HSC-CFU assay according to the prior art (comparative example)

[0101] This assay is similar to the classical methylcellulose CFU assay. In the latter, a total of 500 - 1000 CD34+ cells are seeded in 3 ml of semi - solid medium (or 250 - 500 CD34+ cells / 1.5 ml), and then the medium is aliquoted into two 35 - mm wells (approx. 1 ml / well). From the initial experiments, we observed that the ideal number of seeded CD34+ cells that gives the most reliable output in terms of colony size, colony type, and total number of colonies / well is 250 cells / 1 ml. Based on this observation, and considering that the total volume seeded in a 96 - well plate is 960 μl (96 wells × 10 μl / well) and not all CD34+ cells form colonies, we performed experiments with different cell seeding numbers and we concluded that the ideal seeding is 250 cells / 960 μl, which corresponds to a concentration of 2.5 cells / well. In fact, the results obtained by this method yield a similar number of colonies between the semi - solid - based CFU assay in 35 - mm dishes ("classical" CFU assay) and the liquid - based CFU assay in 96 - well plates ( Figure 4 ). Thus, the correlation is: 250 cells / 35 - mm dish corresponds to approximately 250 cells / 96 - well plate. A total of three 96 - well plates are seeded per sample.

[0102] Figure 4 Shows the comparison of the average total colony counts found in two 35 mm dishes and the average total colony counts found in three 96-well plates when inoculated with the same cell concentration. Error bars represent standard deviation.

[0103] The cumulative results of a total of 6 experiments are also illustrated in Table 2, and show that the 96-well data is similar to the 35 mm dish data. All measured p-values are higher than 0.05 (two-tailed t-test), indicating no statistical difference between the two assays in terms of colony counts and colony type formation.

[0104] Type 35 mm dish 96-well plate P value BFU-E 33.3 30.0 0.27401122 CFU-G 23.8 23.8 0.98668047 CFU-M 5.8 6.3 0.60714795 CFU-GM 3.1 1.9 0.06952218 CFU-GEMM 0.8 0.5 0.28543638 Total number of colonies 66.8 62.4 0.50895526

[0105] Table 2 Cumulative data

[0106] Immunofluorescence staining with HSC-CFU antibody mixture and subsequent data analysis

[0107] CD34+ cells from buffy coat were cultured in HSC-CFU assay medium for 14 days, stained with the HSC-CFU antibody mixture as described above and analyzed on a MACSQuant Analyzer 10. The analysis procedure is described as follows:

[0108] 1. Initial flow cytometry analysis was performed by selecting red colonies with CD235a positive signal (e.g., BFU-E colonies).

[0109] 2. Gates were drawn to include all events ( Figure 5 a).

[0110] 3. Next, all doublets were excluded by gating on single cells in the plot of FSC-A versus FSC-H ( Figure 5 b).

[0111] 4. All single cells were shown in two new plots: for the first ( Figure 5 c), the y-axis was adjusted to CD15-APC and the x-axis was adjusted to CD235a-PE. For the second ( Figure 5 d), the y-axis was adjusted to CD15-APC and the x-axis was adjusted to CD14Vioblue. The quadrants for dividing the populations were set as shown ( Figure 5 c and 5d).

[0112] 5. Name the target regions: Add names to Figure 5 the CD235a-PE positive region of c, Figure 5 the CD15-APC positive and CD14-VioBlue positive regions of d.

[0113] 6. Apply this analysis template to all remaining wells. Generate information on the percentage of CD14+, CD15+ and CD235a+ cells in each quadrant and output it to an excel worksheet for further computational analysis. Always ensure that the general gating parameters still apply and that no regions shift after the flow cytometer is re-compensated.

[0114] Data analysis

[0115] Flow cytometry analysis will generate specific staining for each well, and thus each well will display distinct positive events in one or more regions corresponding to the respective markers labeled in Figure 5 . Based on these markers, each colony can be classified by the percentage of stained cells in each of these regions. According to Figures 6 - 9 and the guidelines summarized in Table 3, colonies are characterized as BFU-E, CFU-GEMM, CFU-M, and CFU-GM.

[0116] BFU-E colonies can be determined by the number of positive CD235a events they exhibit. More than 50% of the total events must be positive for CD235a-PE. Figure 6 Showing BFU-E characterization. BFU-E colonies are positive for the erythroid marker CD235a.

[0117] When more than 50% of the total events are positive for CD15-APC, the colony is CFU-G. Figure 7 Showing CFU-G characterization. CFU-G colonies are negative for the erythroid marker CD235a and express CD15 in more than 50% of the cells.

[0118] Cells belonging to CFU-M colonies are positive for CD14 for more than 50% of the total events. Figure 8 Showing CFU-M characterization. CFU-M colonies should be at least 50% positive for CD14.

[0119] CFU-GM colonies contain progenitors of the granulocyte and monocyte / macrophage lineages. Therefore, staining in the CD14-VioBlue positive and CD15-APC positive regions must exceed 30% for each of these regions. The colony should also be negative for the erythroid marker CD235a. Figure 9 Illustrating CFU-GM characterization. When it expresses more than 30% CD14 positive cells and more than 30% CD15 positive cells, the colony can be characterized as CFU-GM. It should also be negative for the erythroid marker CD235a. The nature of CFU-GEMM is cells that contain all bone marrow progenitors and are also positive for the expression of the erythroid marker CD235a.

[0120] CFU-GEMM colonies are multi-lineage progenitor cells that contain cells of all other bone marrow progenitor cells. The criteria that CFU-GEMM must meet are that CD235a-PE positive events must exceed 20% of the total cells, CD15-APC positive events must exceed 15% of the total cells, and simultaneously CD14-VioBlue positive events must exceed 15% of the total cells.

[0121] In summary, based on the specific staining of each well, the detection parameters listed in Table 3 can be used to identify colonies. For example, any well showing at least 15% CD15 positive, 14% CD14 positive, and 20% CD235a positive events can be a CFU-GEMM colony.

[0122] The hematopoietic system is constantly self-renewing and includes cells at different stages of maturation. These include rare primitive stem cells with multi-lineage differentiation ability and high self-renewal, as well as progenitor cells with limited differentiation and self-renewal potential. Currently, semi-solid media have become the standard for counting and evaluating stem cells and progenitor cells as colony-forming units (CFU). Based on methylcellulose in IMDM supplemented with fetal bovine serum (FBS) and different growth factors, these media mimic the role of stromal cells and provide optimal growth conditions. As previously mentioned, after the incubation period, colonies are evaluated by visual observation under an optical microscope. However, the results obtained in this way are biased and error-prone because they depend entirely on the expertise of the researcher responsible for the assay. One way to simplify the analysis of the CFU assay while still obtaining information about lineage-specific progenitor cell growth is to perform the assay in lineage-specific media, where all colonies are derived from one progenitor cell subtype, such as only CFU-GM or BFU-E, etc. However, this type of culture does not represent the entire pool of progenitor / stem cells and does not create a basis for robust evaluation. Therefore, the only way to address the low reproducibility problem in visual colony evaluation and increase robustness and eliminate any bias is to develop methods for systematic analysis of colonies that combine differentiation in cell culture with a user-independent readout assay.

[0123] To this end, we have developed a method according to the present invention, which provides a standardized method for analyzing hematopoietic stem cells and progenitor cells, because it combines differentiation in cell culture, followed by flow cytometry-based evaluation, and thus eliminates the need for user-dependent visual scoring under a microscope.

[0124] After colony formation, the colonies are analyzed by flow cytometry and evaluated according to their type after staining with the HSC-CFU antibody mixture. Thus, each colony can be easily identified by the corresponding marker combination. In addition, based on the frequency of each colony type, the percentage of occurrence of each colony can be determined relative to the total number of colonies. The innovation of the assays and methods according to the invention lies in the methylcellulose-free HSC-CFU medium and the simultaneous combination with the HSC-CFU antibody mixture, since the assays and methods allow the differentiation of dry / progenitor cells and provide a comprehensive characterization of each colony based on the specific staining presented by them. This setup provides a standardized, user-independent analysis of the HSC-CFU assay, allows automation in combination with any flow cytometer, and ultimately provides reproducible and consistent results.

Claims

1. A method for detecting differentiated hematopoietic stem cells, comprising the following steps: a) isolating undifferentiated hematopoietic stem cells in groups of 1 - 1000 cells on a support; b) proliferating the isolated cells by providing a cell culture medium containing growth factors to form cell colonies of differentiated hematopoietic stem cells; c) contacting the cell colonies with one or more labeled conjugates, the labeled conjugates comprising at least one detection moiety and at least one antigen recognition moiety directed against CD14, CD235a or CD15; d) detecting the relative amount of differentiated hematopoietic stem cells in the cell colonies labeled with the labeled conjugate.

2. The method according to claim 1, characterized in that The method is carried out in the absence of methylcellulose.

3. The method according to claim 1 or 2, characterized in that A cell sample containing undifferentiated hematopoietic stem cells is provided to step a), and red blood cells are lysed in the cell sample.

4. The method according to claim 1 or 2, characterized in that A cell sample containing undifferentiated hematopoietic stem cells is provided to step a), and CD34+ cells are enriched in the cell sample.

5. The method according to claim 4, characterized in that The CD34+ cells of the cell sample are enriched to a purity of at least 50%.

6. The method according to claim 1 or 2, characterized in that, The detection moiety is selected from a chromophore moiety, a fluorescent moiety, a phosphorescent moiety, a luminescent moiety, an absorptive moiety, a radioactive moiety, a transition metal and an isotope mass tag moiety.

7. The method according to claim 1 or 2, wherein the antigen recognition moiety is an antibody, a fragmented antibody, a fragmented antibody derivative, a peptide / MHC complex targeting a TCR molecule, a cell adhesion receptor molecule, a receptor of a co - stimulatory molecule or an artificially engineered binding molecule.

8. Use of the method according to any one of claims 1 - 7 for determining the differentiation status of stem cells in a cell sample.

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