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Jackie Damen (Senior Scientific Advisor, Hematopoiesis): The hematopoietic CD34⁺ niche is found in the bone marrow. Niches are local tissue microenvironments that maintain and regulate stem cells. After birth, hematopoietic stem cells (HSCs) are found in a niche that starts in the long bone marrow cavities. Here, non-hematopoietic cells, including osteoblasts and marrow stromal cells, have been have been found to produce hematopoietic cytokines that support primitive haematopoiesis. For more information, refer to the review by Sean Morrison and David Scadden.

Daniela Mora Ortiz, MD (Scientific Operations Associate & Lab Manager): Peripheral blood cell count differs slightly in the different age groups. At birth, the red blood cell (RBC) count is significantly higher compared to in early childhood and adults. The number declines within the first week of life since the lifespan of these erythroid cells is shorter. The counts fluctuate and stabilize at the end of the neonatal stage (birth - 28 days old).

The number of circulating CD34⁺ cells is higher closer to birth and then declines. At around 2 - 4 years old there is an abrupt change in HSC proliferative activity. This has been inferred from measurements of the rate of decline in telomere length of circulating leukocytes but overall, the hematopoietic system is modestly affected by aging until after the age of 65. The proliferative capacity of the marrow cells from older adults can sustain normal blood cell counts, but the reserve capacity may be limited, as observed by decreased responses to G-CSF with regards to mobilization of neutrophils and the response of CD34⁺ cells in older patients. In addition, marrow cellularity decreases with aging.

Dr. Jackie Damen: Yes, for cell therapy products used in gene therapies and more-than-minimally-manipulated cell therapies in prolonged cultures, regulations specify sterility testing for bacteria and fungi using microbial testing methods equivalent to or better than those described in 21 CFR 610.12. Federal regulations (21 CFR 1271: current Good Tissue Practices) require the collection, processing, packaging, and distribution of all cellular therapeutics by methods that prevent the introduction, transmission, or spread of communicable diseases, including viral, bacterial, fungal, and/or parasitic infections.

Dr. Jackie Damen: Investigation of the expansion of CD34⁺ cells ex vivo started in the 1990s and continues today. One breakthrough in the ex vivo culture of CD34⁺ cells was the discovery that bone marrow stromal cells and stromal cell lines in CD34⁺ culture could be replaced by serum-free media formulations, when combined with cytokines including SCF, Flt3,TPO, IL-3, and IL-6 in suspension cultures. Recently, high-throughput screening has identified new molecules; prostaglandin E2 (PGE), StemRegenin 1 (SR1), and UM171, that improve the frequency of the symmetric self-renewing HSC. Addition of these molecules results in higher numbers of the engraftable CD34⁺ cell compared to cultures with added cytokines alone.

Leon Lin, PhD (Scientist, Research and Development): Yes, we recommend using UM729 for expansion of leukemic stem and progenitor cells. We have in-house data showing that UM729 at 1 μM performs similarly to UM171 at 175 nM in terms of maintenance and expansion of CD34⁺ and CD34⁺CD90⁺CD45RA^- cells. The Sauvageau lab's original small molecule screen identified UM729 as capable of expanding acute myeloid leukemia (AML) leukemic stem and progenitor cells ex vivo (Pabst et al., 2014). Their more recent publication also utilized UM729 to culture AML cells to perform drug candidate screening (Baccelli et al., 2017).

Dr. Jackie Damen: To identify lineage differentiation, flow cytometry is typically used after staining with a lineage-specific marker. For human erythrocytes, these would be expected to be positive for both CD71 and GlyA. However, if you are distinguishing different lineages using the the CFU assay, for colonies from myelopoiesis there are 3 main colony types: myeloid (CFU-GM), erythroid (BFU-E and CFU-E), and multi-potential progenitors (CFU-GEMM).

Once the progenitors grow and mature in an optimized formulation of �ѱ�ٳ�ǰ�ܱ���™ media, the result is colonies that contain mature cells that differ in size and color. Erythroid progenitors (BFU-E and CFU-E) will consist of small, red colored erythroblast cells at the edges of the colony and are distinctively different (when observed down the 10x objective of an inverted microscope) from myeloid colonies (CFU-GM), which consist of larger, clear and shiny granulocytes, monocytes, and macrophages.

The identity of mature cells within colonies from the various lineage types was confirmed historically using histological hematoxylin and eosin (H&E) staining as well as evaluation of lineage-specific surface marker expression using FACS.

For more, refer to the Atlas of Hematopoietic Colonies from Cord Blood.

Dr. Jackie Damen: From publications reviewed in the webinar, the recommendation is to ensure mobilized PB and all samples of HSPC are stored at 4°C. The data from Janzen et al. (2009) showed cell viability and potency could be maintained for 48 hours, especially if the cell density is not excessively high (>50 million/mL). It is important to ensure that during shipping the cells do not come in contact with frozen ice packs. Depending on the downstream application, 24 hours post collection can be used as a cutoff to ensure good quality cells for additional processing steps.

Dr. Jackie Damen: The CD34⁺ cells derived from pluripotent stem cell (PSC) cultures are not functionally the same as the CD34⁺ cells derived from adult sources. Depending on the stage of embryogenesis from which the cells arise, their function at early stages of hematopoiesis are different. For example, lymphopoiesis is not required at primitive stages of development.

Depending on the method used to derive HSPCs from PSC cultures the total CFU frequency and lineage progenitor frequency can be very different. Most research has shown that the frequency of CFU/CD34 input is lower for PSC derived CD34⁺ cells (compared to both BM and mPB sources) and not all lineages are represented. In addition, the resulting colonies are typically smaller and tend to have fewer cells per colony since the progenitors do not seem to mature to the same extent as adult-derived CD34 cells in CFU cultures.

Dr. Jackie Damen: Viruses used as vectors in cell and gene therapy are replication deficient and therefore cannot cause infection. Gene therapy approaches using viruses to infect hematopoietic stem cells as a treatment for hematological genetic disorders (like sickle cell disease and thalassemia) started in the early 1990s. Since then, various types of viral vectors have been evaluated and are replication deficient; however, their use in clinical trials using gene therapy have not been without risks resulting from immune responses, insertional mutagenesis, viral tropism, and off-target outcomes.

Dr. Jackie Damen: The CFU assay provides retrospective information on the functional viability and potency of hematopoietic cell therapy products (HCTPs) as part of a QC test. Clinical labs may not count the specific numbers of colonies, instead reporting either ‘growth’ or ‘no growth’ as the result of the CFU assay. This would confirm that cells were still functionally viable at the time of injection, however it will not be known if patient engraftment has been successful until 2 - 4 weeks post transplant.

Processing labs, however, may define a specific plating concentration. In this case, the CFU growth or frequency that is reported indicates the minimum number of CFUs required for transplant, and is based on pre-freeze data. In addition, a review by Mike Watts, David Linch, and colleagues (2016) outlines results from a previous publication that shows neutrophil recovery within 14 days in >99% of patients (and none had a delayed engraftment beyond 28 days) when a CFU-GM dose of at least 2 x 10^5/kg was used so knowing this number can be informative. So, despite being retrospective, a minimum plating concentration of thawed cells in the CFU assay can confirm the dose and quality of the cells when neutrophil recovery in patients takes longer than 14 days.

Dr. Jackie Damen: The Long-Term Culture Initiating Cell (LTC-IC) assay uses the CFU assay as a readout but starts with co-culture of the earliest HSC on stromal cells, or stromal cell lines, without the additional requirement for cytokines. That said, present day protocols for this assay use a stromal cell line engineered to express critical human cytokines to make the assay more robust.

The LTC-IC assay enables measurement of the frequency of early HSCs in a test population following addition of this non-adherent cell population into cultures that have irradiated stromal cells. The protocol uses a limiting dilution strategy, requiring the initiation of replicate cultures (typically >12) of 4 - 6 cell dilutions of the test cell population. The cultures are then maintained for more than five weeks, with weekly half-media changes. At the end of the culture period all the non-adherent and adherent cells are harvested and plated into a CFU assay. Each replicate is plated into one CFU assay and after 14 days, the assays are evaluated as positive or negative for growth of progenitors. The frequency of negative replicates at each cell dilution can then be calculated and Poisson statistics used to determine the frequency of HSCs.

In theory, the LTC-IC assay would be expected to be more predictive of the in vivo engraftment assay. However it is a difficult assay and therefore not very robust—although there are publications from the 1990s that used this assay in lieu of the engraftment assay, which was developed later. Now, mostly because of improvements and the availability of new NSG mouse strains, the LTC-IC assay is not performed as much and does not appear in more recent publications in the field.

Dr. Jackie Damen: The granulocytic lineage consists of three mature cell types: neutrophils, basophils, and eosinophils. The only way to truly identify the frequency of these in peripheral blood is based on histological staining with hematoxylin and eosin (H&E) and evaluation of relevant morphological features that distinguishes these three types based on their size, color, and shape of the nucleus.

In a CFU assay, one only has the size of the mature cells to help identify the progenitor type and cannot visualize the shape of the nuclei. The shape of the nuclei is key in determining the difference between, for example, granulocytes and monocytes. The only way to identify the composition of the cells within a CFU-GM colony is to pluck the colony and evaluate the mature cells within the colony following a cytospin and subsequent H&E staining. However, most colonies are a heterogeneous mix of immature and mature cells that represents the lineage potential of the progenitor.

Alternatively, cells plucked from individual colonies can be stained with cell surface markers that are characteristic for the various mature cell types. However, this approach often involves staining of multiple surface markers to be definitive. Historically, histological staining, cell-surface expression by flow cytometry, and cytokine requirements have all been used to confirm lineage specific progenitor types.

Dr. Jackie Damen: Despite some early publications in the 1980's that suggested CFU cultures could be shown for T cells harvested from peripheral blood and the bone marrow, it was realized that growth and differentiation of lymphoid progenitors was limited to early pro-T and pro-B colonies only. The identification of Notch signaling as a crucial mediator for lymphopoiesis led to dramatically improved methods and expansion on the mouse bone marrow-derived stromal cell OP9 engineered to overexpress Notch ligand. This has greatly improved the ability to expand and derive mature human T cell subsets for therapeutic applications.

Dr. Jackie Damen:If the objective is to obtain T cell progenitors, a CFU assay is only necessary to qualify the quality of the starting sample. Since the final product in this case is not expected to reconstitute the hematopoietic system in a compromised patient, the stability of frozen T cell products would not require the CFU assay. For T cell products, a different potency assay to identify T cell quality and function would be required. If, however, you are working on the production of engineered T cells to target and eliminate AML, then the CFU assay can be used to ensure there are no off-target effects of the T cells on CD34⁺ cells that could result in cytopenia.

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Dr. Jackie Damen: There are a few examples of results from the CFU assays that correlate with good engraftment. Specifically, in a 2014 publication from Dr. Guy Sauvageau's lab (Fares et al, 2014), it was shown that when expanding cord blood CD34⁺ cells in the presence of UM171, there was an increase in the CD34⁺CD90⁺ population as well as in the numbers of the multipotential CFU-GEMM colonies. These two parameters correlated with an improvement in the frequency of HSCs, as determined by engraftment in the SCID repopulating cells (SRC) assay.

In addition, there seems to be good anecdotal data for this correlation: preliminary results presented in abstracts at meetings like ASH have shown trends in CFU frequency and colony size with engraftment potential and chimerism. Several translational studies have also concluded that the CFU-GM assay strongly correlates with successful patient recovery following a transplant.

We know also that when we expand CD34⁺ cells in culture, especially without the use of small molecules that support the self-renewing HSC divisions, and remove cells after 5 - 7 days to plate these in the CFU assay, two things are certain: the frequency of CFUs drop and colonies become smaller as their proliferative potential is exhausted. When there are no more progenitors that can be read out in the CFU assay, the expectation is that there would also be no cells capable of long term engraftment.

Dr. Jackie Damen: The use of trypan blue to evaluate cell viability has been used in cell culture for many years and can be performed manually (using a hemocytometer) or using automated cell counters. Viability determined manually with trypan blue is more subjective, based in part on the fact it is an exclusion dye compared to nuclear dyes like 7AAD that are more sensitive and robust. There is no consensus in the field to recommend a specific viability dye, but it is understood that trypan blue typically results in higher viability assessment when compared directly to 7AAD and AO/PI. Different viability stains can be used and validation with additional assays to confirm functional potency is typically required.

As far as a recommendation, I prefer using AO/PI staining. When we switched in the lab from using trypan blue to AO/PI, we realized that our viability for qualified lots of BM MNCs were in general ~10-15% lower but our CFU output was ~10 - 15% higher due to improved sensitivity of AO/PI compared to trypan blue.

Dr. Jackie Damen: The seeding density is dependent on the cell source and species when plating in the CFU Assay. For human samples, this is dependent on the frequency of CD34⁺ expected in these samples. Since this can vary between donors, we recommend ranges and plating 2 - 3 cell concentrations within the ranges (provided in the webinar on slide 27 and listed below) to ensure the CFU frequency is within the linear range and detection limits of the CFU assay.

Dr. Jackie Damen: Optimal combinations and cytokine concentrations have been the subject of many investigations and attempts to standardize the CFU assay over the last 30 years. For human CFUs, most media formulations require appropriately qualified FBS with cytokine combinations that are optimal for the growth and differentiation of each progenitor type.

To visualize all the potential progenitors in a single patient sample for myelopoiesis, cytokine combinations that support the simultaneous growth of erythroid and myeloid progenitors have included recombinant human cytokines for SCF, GM-CSF, EPO, G-CSF, IL-3 and, in some cases, IL-6 if CD34⁺ are first purified.

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Dr. Jackie Damen: Yes, the scoring of CFU assays can be subjective. Some of the most common and particularly challenging phenomena encountered when scoring CFU colonies is the presence of colonies with multiple foci or clusters, which can be scored as separate colonies and thus erroneously skew the total counts to higher CFU numbers. This, in turn, may lead to an overestimation of HSPC graft potency. Conversely, high plating concentrations can result in overlapping and underdeveloped colonies that can lead to under-scoring of CFUs. Training has been shown to improve consistency between manual counters with regard to both identification of colony number as well as lineage identification of each colony type.

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Dr. Jackie Damen: Validation studies to compare manual versus automated CFU counting have been investigated by various groups as part of their qualification process when switching from manual to automated CFU scoring. Internal studies carried out at �����ƽ�� show a strong correlation between manual and automated CFU scoring. These studies were carried out by comparing manual counts (performed by 3 to 7 experienced counters) of colony assays using cord blood, bone marrow, or mobilized peripheral blood, and comparing these counts to automated counts on a ���շ��ѱ��������Dz�™ instrument. Results showed an r value of 0.96 for total colonies, indicating a strong correlation between manual and automated CFU scoring.

More recently, Velier M et al. (2019) published that automated scoring using ���շ��ѱ��������Dz�™ reduces the coefficients of variation of repeatability, inter-operator variability, and intermediate precision.

Dr. Jackie Damen: When validating automated scoring, three errors can be identified: false positives (resulting from scratches or artifacts in the media and cultureware or overlapping colonies), false negatives (colonies not scored due to merging of colonies or colonies missed on the edges of the wells), and misidentified colonies (colonies that are scored as the wrong lineage).

Independent customer studies that have been shared with us have shown that correction of these errors, using the images available from colonies scored using ���շ��ѱ��������Dz�™ algorithms, resulted in data that was not statistically different. Differences in colony numbers and identification between corrected and uncorrected images were not significant. Similarly, the variability in counts from corrected and uncorrected images were shown to have similar or lower coefficients of variation (CVs) when using automated scoring compared to manual counting.

Find out more in the .

Dr. Jackie Damen: Imaging with ���շ��ѱ��������Dz�™ is key to the way the ���շ��ѱ��������Dz�™ algorithm works and the algorithm cannot be used seperately to score images of colonies taken with a digital camera as the entire image is required for scoring with the ���շ��ѱ��������Dz�™ algorithms.

Amanda Fentiman, MSc (Product Manager, Hematology): We offer complete media for CFU assays, �ѱ�ٳ�ǰ�ܱ���™, which consists of methylcellulose media and supplements with prequalified serum, and cytokines optimized for human or mouse hematopoietic colony growth. For human cells our �ѱ�ٳ�ǰ�ܱ���™ H4034 Optimum is the best media for myeloid and erythroid colony growth from blood or bone marrow. We also offer serum-free formulations and cytokine-free versions, depending on what you need.

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Dr. Jackie Damen: There are other international societies, including the Joint Accreditation Committee ISCT-Europe (JACIE), which is an accreditation body for hematopoietic stem cell therapy. Their standards also require a potency assay for cell therapy products undergoing manipulation as well as for stability testing following storage. The CFU assay is suggested in addition to determination of CD34⁺ cell numbers; however, there is no mention of standardized protocols for these assays that I know of. Groups can validate their own assays and protocols and, with regards to those involving FACS, some publications have included the use of ALDH or the use of apoptotic dyes when evaluating viable CD34⁺ to ensure functional viability.