Proliferation/Potency Assays: Is there more to CD34 and CFU?

Transcription

1 Proliferation/Potency Assays: Is there more to CD34 and CFU? CBMTG 2012 Laboratory Committee Meeting April 11, 2012 Toronto, ON, Canada Leah Marquez-Curtis CBS-Edmonton

2 OUTLINE I. Rationale and Definitions II. Organizational hierarchy of the blood-forming system III. Assays to detect hematopoietic stem/progenitor cells (HSPC) Analysis of surface markers (CD34) In vivo assays (CFU-spleen, SCID-repopulation assay) In vitro assays (LTC-IC, CAFC, CFU) IV. Alternative readouts for cell proliferation Intracellular ATP Aldehyde dehydrogenase V. Clinical studies on graft characteristics predicting engraftment VI. Improvements to the CFU assay VII. Summary and Conclusions

3 Rationale Up to 20% of patients receiving CB transplant experience graft failure. Engraftment is often delayed due to a lack of potency of the product, i.e., low cell dose. Adequate assays are needed in order to: 1) ensure that the most suitable and highest quality cellular products are available for clinical transplantation 2) monitor the effect of processing, cryopreservation, and thawing on the quality of stem cell products 3) comply with the standard requirements for accreditation

4 INTERNATIONAL STANDARDS FOR CELLULAR THERAPY PRODUCT COLLECTION, PROCESSING, AND ADMINISTRATION Fifth Edition March 2012 NOTICE D6.1.3 There shall be the establishment of appropriate and validated assays and test procedures for the evaluation of cellular therapy products. D For all cellular therapy products, a total nucleated cell count and viability measurement shall be performed. D For HPC products, a CD34 assay shall be performed. D For cellular therapy products undergoing manipulation that alters the final cell population, a relevant and validated assay, where available, shall be employed for evaluation of the target cell population before and after the processing procedures.

5 Definitions Potency: the therapeutic activity of a product as indicated by appropriate laboratory tests or adequately developed and controlled clinical data Hematopoietic progenitor cells (HPC): A cellular therapy product that contains selfrenewing and/or multi-potent stem cells capable of maturation into any of the hematopoietic lineages.

6 Characteristics and Assays of HSC Self-renewal ability to give rise to identical daughter HSC without differentiation Multipotency ability to produce all of the different cell types found in blood Therefore, potency measurements should reflect these two relevant biological attributes. Cell proliferation or self-renewal: measures the number of cells produced Differentiation potential: estimates the number of different lineages represented in its progeny

7 PHENOTYPE ASSAYS Lin - CD34 + CD90 + CD38 - c-kit + CD133 + Stem Cells SCID Repopulating Cells Long-term culture initiating cells Cobblestone area forming cells Lin + CD34 + CD90 - CD38 + CD33 + Multi-potential Progenitors Lineage-Committed Progenitors CFU assay Granulocyte Macrophage Erythrocyte Megakaryocyte Lin + CD34 - Dendritic cells Monocytes/ Macrophages Morphological stains NK cells T lymphocytes B lymphocytes Granulocytes Red blood cells Mature Blood Cells Platelets From: Bert Wognum, Mini-Review on HSPC, StemCell Technologies

8 Phenotype of human HSC Lineage-negative CD34-positive Thy 1.1 lo (CD90) c-kit-positive CD133-positive CD38-negative Hoescht side population cells SLAM family (CD150-positive); CD201 Molecular markers have been used to characterize HSCs, but these cells are still best defined functionally. Phenotyping by FACS analysis may be considered as a surrogate assay, because there is a generally a good correlation between phenotype and reconstitution potential in vivo.

9 First functional in vivo assay for HSC Colony forming unit spleen (CFU-S): Nodules were observed between 1 to 3 weeks. Their numbers are proportional to the number of BM cells injected. Assays shortterm HPC. (James E. Till and Ernest A. McCulloch, 1961, University of Toronto)

10 SCID mice model SCID: severe combined immune deficiency Characterized by inability to mount immune response Routinely used for transplantation of human cells The more severely compromised ones naturally tend to yield the best engraftment, such as the non-obese diabetic (NOD)- SCID mice with reduced NK activity. Since the first description by John Dick in 1988 (Ontario Cancer Institute, many strains of immune- deficient mice for engraftment of human cells have been developed.

11 Long-term repopulation assays The frequency of repopulating cells is usually determined by limiting dilution analysis (varying doses of test cells compete against a set number of BM cells) and expressed as competitive repopulating units (CRU). A test population is considered to contain LT-HSC when a recipient contains donor-derived cells of all lineages even after at least 16-weeks following transplantation. Serial transplantation assay: most stringent test for stemness ; test cells transplanted, harvested and further transplanted to secondary, tertiary, and so on, recipients

12 SCID-repopulating cell (SRC) Assay Provides a quantitative in vivo assay for primitive human HSC Frequency of SRC in MNC from: CB: 1 in 9.3 x 10 5 ( one in a million ) BM: 1 in 3 x 10 6 mpb: 1 in 6 x 10 6

13 LT-HSC vs. ST-HSC Long-term reconstituting HSC incapable of maintaining survival in the short-term; minimum of 16 weeks is required and an optimal 6 months is suggested for monitoring longterm reconstitution post-transplant Short-term reconstituting hematopoietic stem cells more rapidly reconstitute hematopoiesis towards myeloid lineage -incapable of long-term reconstitution -important for short-term survival (4 wks) Both ST-HSC and LT-HSC populations are necessary for the successful outcome of stem cell transplantation.

14 In vitro stem cell assays Developed by Eaves Terry Fox Lab, BC Long-term culture-initiating cell: represents a cell population that possesses considerable selfrenewal ability; characterized by Lin - CD34 + CD38 - Cobblestone area-forming cells: a variant of the LTC-IC assay; shows good correlation with different hematopoietic subsets as tested with other assays (CFU) These assays are very labour-intensive. Sutherland, Lansdorp, Henkelman, Eaves and Eaves PNAS USA; 87:3584 Udomsakdi, Lansdorp, Hogge, Reid, Eaves and Eaves Blood;80:2513

15 LTC-IC and CAFC assays for HSC

16 Short-term in vitro assay: colony-forming unit (CFU) assay Step 1 Prepare cells Step 2 Add cells to semi- solid culture media (methyl cellulose /IMDM/ FCS/growth factors) Step 3 Plate cell suspension in culture dish and incubate at 37 o C (5% CO 2 ) for 14 days Step 4 Count colonies. Each multipotent or committed progenitor cell present in original sample will give rise to a specific colony e.g. CFU-GM, BFU-E, GEMM, etc.

17 Comparison between in vitro and in vivo stem cell assays Assay Phenotyping (Flow cytometry) (CD34) In vitro clonogenic growth (CAFC, LTC-IC) Transplantation into SCID mice Measures proliferative potential Measures in vivo reconstitution capability Advantages Disadvatages No No Fast Not a functional assay; phenotype changes with exptal conditions and independently of cell function Yes No Allows quantification of different subsets Yes Yes Clinically most relevant Labor-intensive; subjective read-out Slow; expensive In vitro CFU assay No No Quantifies lineagecommitted cells Slow; subjective readout; does not identify more primitive LT-HSC

18 Why Count Colonies, When an Instrument can do the Work for You? Determines the number of viable cells in proliferation Transforms the subjective CFU assay into an instrumentbased quantitative assay Uses an absorbance ELISA plate reader Results obtained in 7 days Easier and cheaper than methylcellulose-based assay Incorporates suspension expansion culture Applicable for bone marrow, cord blood or mobilized peripheral blood

19 It is not necessary to distinguish colony types in order to measure the total CFU content of a graft. Colonies grown in MethoCult Express are larger than in MethoCult media and can be counted as early as after 7 days of culture vs days. This faster and easier CFU assay may improve reproducibility and standardization within and between labs. 7 days 14 days

20 STEMpredict The most rapid assay to predict viability, growth and cellular functionality of stem cells in mobilized peripheral blood, umbilical cord blood and bone marrow products manufactured in a stem cell processing laboratory. Rapid assessment (2-3 days) of HSC viability, growth and cellular functionality in a single assay Allows for assay calibration, standardization and validation Incorporates bioluminomics technology for highly sensitive, fast and reliable results

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22 HALO (ATP) assay performed in parallel with CFU testing of HPC apheresis products All HPC samples routinely diluted to same cell concentration 1/10 diln - Methocult H CD34 + cells/plate DF CD34 + cells/ml Incubate together (37 C humidified CO 2 incubator) HALO: 7 days CFU: days DF1 15x10 6 WBCs/mL 1/10 diln - HALO Master Mix WS1 5x10 5 WBCs/mL 5000 WBCs/well Letcher B et al. CBMTG 2012 Poster Presentation

23 Correlation of HALO results and parallel CFU assay HALO PCA-eq, Mean ATP (um) R 2 = n = # of CFUs seeded per well

24 39 of 49 samples show a better correlation between ATP and CFU

25 Low ATP results did not correspond with CFU counts and ST-engraftment 10/49 samples produced very low ATP results less than 0.04 µm ATP presumed DEAD These samples looked good by CFU assay using the cytokine-matched Methocult media Routine monitoring of short-term engraftment has detected no problems relating to the transplantation of these stem cell products

26 Aldehyde dehydrogenase (ALDH): enzyme activity that measures viable cells Aldefluor fluorescent reagent that stains positive for human hematopoietic stem/progenitor cells (CD34 and CD133) CFU and ALDH br define a high quality CB (Kurtzberg, 2011) Fresh Cord Blood Cells (n=1620) Number of cells containing 1 CFU TNC 132 MNC 48 CD ALDH br 1.2 CD34 +/ ALDH br 0.98

27 Typical FACS plot for HSC from cord blood

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29 Most accurate method to assess graft quality is by analyzing the engraftment after transplant Clinical studies: 1) Migliaccio, Rubinstein (Blood 2000;96:2717) Engraftment data from 204 patients showed that total colony-forming cell (CFC) count, rather than total nucleated cell count (TNC) more rigorously correlated with neutrophil and platelet engraftment and posttransplantation survival 2) Iori et al. (Bone Marrow Transplant 2004;33:1097) Data from 42 leukemia patients who received CB transplant shows that CFU-GM was most important factor for overall survival and is the only factor that affected event-free survival. Conclusion: CFU should be the main parameter in selecting CB units for transplant.

30 Continuation of clinical studies. 3) Yoo et al. (Bone Marrow Transplant 2007;39:515) Data from 53 pediatric patients (<18 years old) showed that post-thaw CFU-GM, TNC and CD34 correlated with the speed of neutrophil engraftment (p=0.004, 037 and 0.004, respectively) whereas only CFU-GM correlated with platelet engraftment (p=0.024). 4) Prasad, Kurtzberg (Blood 2008;112:2979) Unrelated donor umbilical CB transplantation for inherited metabolic disorders in 159 pediatric patients from a single center (Duke University Medical Center, Durham, NC) Among the parameters: total nucleated cells, viability, CD34 and CFU, the infused (post-thaw) CFUs is the graft characteristic that best correlated with the engraftment of both neutrophils and platelets as well as overall survival.

31 Continuation of clinical studies. 5) Page, Kurtzberg (Biol Blood Marrow Transplant 2011;17:1362 Carolinas Cord Blood Bank (Duke University Medical Center) Has transplanted more than 900 patients with CB Retrospective study of 435 patients ( ) TNC, CD34 count and total CFU content evaluated before cryopreservation and after thawing In the post-thaw measurements, CFU dose best predicted neutrophil and platelet engraftment (both p<0.0001); TNC and CD34 counts were only weakly predictive. However, the CFU assay is difficult to standardize and variability within and among laboratories remains.

32 STEMvision Automated Colony-Forming Cell Assay Reader Reduces variability with scoring colonies Saves time (imaging takes just 30 sec/well) Acquires high resolution digital images for archiving and review

33 Regular 35 mm dish Designed to reduce the formation of a meniscus, permitting more uniform distribution of medium and colony distribution. SmartDish TM Results in less optical distortion and crowding of colonies at the edge of the dish and more accurate counts.

34 Summary Potency of stem cell product can be demonstrated by: Direct measurement of specific biological activity (e.g. engraftment and bone marrow reconstitution) Indirect measurement of surrogate characteristics that correlates with the intended function in the clinical setting (CD34 and CFU) Pending further recommendations, the existing surrogate parameters CD34 and CFU provide an acceptable measure of engraftment potential.

35 Correlation between CD34 and CFU in cord blood units y = x R² = CFU (x10e5/unit) CD34 (x10e6) Data from Alberta Cord Blood Bank

36 Conclusions Under normal conditions, correlations between phenotype (flow cytometry), in vitro clonogenic activity and in vivo function are generally good. Conflicting results do occur as when cells express HSC characteristics in one assay and not in another. However, based on retrospective assessment of clinical engraftment, it appears that CD34 and CFU would define a good quality of a cellular product as required by regulatory agencies. Automation and standardization of the CFU assay will result in increased accuracy and reproducibility as well as savings for high-throughput labs.

37 Acknowledgments Canadian Blood Services (CBS), Research and Development Dr. Anna Janowska-Wieczorek Stem Cell Laboratory, CBS, Edmonton Centre Dr. Locksley McGann Brenda Letcher Maria Cabuhat Kelly Murphy Alberta Cord Blood Bank Dr. John Akabutu Sally Shahi

38 Cell Cycle Resting/ Quiescence

39 Found: the mother of all blood cells: CD49f marks the ultimate HSC Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment Science 8 July 2011: [DOI: /science ]

40 Comparison of human STRC and LTRC Property STRC-Myeloid STRC- Lymphoid LTRC Time of peak engraftment (weeks posttransplant) 3 weeks 6 to 8 weeks 8 to 16 weeks Progeny lineages Erythroid and Megakaryocytic B-lymphoid and Granulocytic B-lymphoid and Granulocytic Phenotype CD34 + CD38 + CD34 + CD38 - CD34 + CD38 - Ability to engraft NOD-SCID mice No No Yes

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