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

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

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

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

D6.1.3 There shall be the establishment of appropriate and validated assays and test procedures for the evaluation of cellular therapy products.

D6.1.3.1 For all cellular therapy products, a total nucleated cell count

and viability measurement shall be performed.

D6.1.3.2 For HPC products, a CD34 assay shall be performed.

D6.1.3.3 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. INTERNATIONAL STANDARDS FOR CELLULAR THERAPY PRODUCT COLLECTION,

PROCESSING, AND ADMINISTRATION

Fifth Edition March 2012

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

self-renewing and/or multi-potent stem cells

capable of maturation into any of the

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

Multi-potential Progenitors Stem Cells

From: Bert Wognum, Mini-Review on HSPC, StemCell Technologies

Lineage-Committed Progenitors

Granulocytes Dendritic cells

NK cells

Red blood cells Platelets B lymphocytes

Monocytes/ Macrophages

T lymphocytes

Mature Blood Cells

ASSAYS

SCID Repopulating Cells

Long-term culture initiating cells Cobblestone area forming cells

CFU assay Granulocyte Macrophage Erythrocyte Megakaryocyte Morphological stains PHENOTYPE Lin- _CD34+ CD90+ _CD38- c-kit+ CD133+ Lin+ _CD34+ CD90- _CD38+ CD33+ Lin+ _CD34

-Phenotype of human HSC

• 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.

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 short-term HPC.

(James E. Till and Ernest A. McCulloch, 1961,

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.

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

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

(“one in a million”)

BM: 1 in 3 x 10

mPB: 1 in 6 x 10

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 long-term 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

In vitro stem cell assays

Developed by Eaves group @ Terry Fox Lab, BC

• Long-term culture-initiating cell: represents a cell

population that possesses considerable

self-renewal 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. 1990. PNAS USA; 87:3584 Udomsakdi, Lansdorp, Hogge, Reid, Eaves and Eaves. 1992. Blood;80:2513

LTC-IC and CAFC

assays for HSC

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 37o_{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.

Assay Measures proliferative potential Measures in vivo reconstitution capability Advantages Disadvatages Phenotyping (Flow cytometry) (CD34)

No No Fast Not a functional assay; phenotype changes with exptal conditions and independently of cell function In vitro clonogenic growth (CAFC, LTC-IC) Yes No Allows quantification of different subsets Labor-intensive; subjective read-out Transplantation into SCID mice

Yes Yes Clinically most relevant Slow; expensive In vitro CFU assay No No Quantifies lineage-committed cells

Slow; subjective read-out; does not identify more primitive LT-HSC

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 instrument-based 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

7 days 14 days

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. 14 - 16 days.

This faster and easier CFU assay may improve reproducibility and standardization within and between labs.

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

HALO (ATP) assay performed in parallel with

CFU testing of HPC apheresis products

Incubate together (37°C humidified CO₂ incubator) HALO: 7 days CFU: 14-16 days DF1 15x106 _WBCs/mL WS1 5x105 _WBCs/mL All HPC samples routinely diluted to same cell concentration DF2 5000 CD34+ _cells/mL 1/10 diln - Methocult H4434 1/10 diln - HALO Master Mix 500 CD34+ _cells/plate 5000 WBCs/well

Correlation of HALO results and

parallel CFU assay

R2 _{= 0.2176} 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 20 40 60 80 100 HAL O P CA -e q, M e a n A TP ( uM )

# of CFUs seeded per well

39 of 49 samples show a better

correlation between ATP and CFU

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

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 ALDHbr _{define a high quality CB (Kurtzberg, 2011)}

Fresh Cord Blood Cells (n=1620)

Number of cells containing 1 CFU TNC 132 MNC 48 CD34+ 2.5 ALDHbr 1.2 CD34+/ALDHbr 0.98

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 post-transplantation 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.

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.

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 (2000-2008)

• 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

STEMvision™

Automated Colony-Forming Cell Assay Reader

Reduces variability with scoring colonies Saves time (imaging takes just 30 sec/well)

Regular 35 mm dish

SmartDishTM

Designed to reduce the formation of a meniscus, permitting more uniform distribution of medium and colony distribution.

Results in less optical distortion and crowding of colonies at the edge of the dish and more accurate counts.

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.

y = 4.4028x + 4.5761 R² = 0.6756 0 10 20 30 40 50 60 70 0 2 4 6 8 10 12 C F U ( x10e5/ u n it ) CD34 (x10e6)

Correlation between CD34 and CFU in cord blood units

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.

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

Cell Cycle

Resting/ Quiescence

Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment

Science 8 July 2011: 218-221. [DOI:10.1126/science.1201219]

Comparison of human STRC and LTRC

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