Jackie Damen, PhD (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 (Scientist, Scientific Support, Epithelial Cell Biology): 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 MethoCult™ 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.

Learn more about the LTC-IC assay >

Colin Hammond, PhD (Scientist, Research and Development): The CFU assay can also be used to assess the ability of expanded or genetically modified HSPCs to form colonies. The efficiency of expanded or genetically modified cells to form colonies in the CFU assay will depend on the conditions of the expansion, including culture time and components of the culture medium, as well as the specifics of any editing procedure and genetic target. Ranges of plating densities should be used for these samples to ensure a scorable colony density is obtained.

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.