Rapid potency assessment of autologous peripheral blood stem cells by intracellular flow cytometry: the PBSC-IL-3-pSTAT5 assay.

BACKGROUND AIMS: The current gold standard for stem cell product potency assessment, the colony-forming unit (CFU) assay, delivers results that are difficult to standardize and requires a substantial amount of time (up to 14 days) for cellular growth. Recently, the authors developed a rapid (<24 h) flow cytometry assay based on the measurement of intracellular phosphorylated STAT5 (pSTAT5) in CD34+ cord blood stem and progenitor cells in response to IL-3 stimulation. The present work presents a novel adaptation of the protocol for use with autologous peripheral blood stem cells (PBSCs) and a performance comparison with the CFU assay. METHODS: The flow cytometry intracellular staining assay was optimized for PBSCs, and patient samples were analyzed using the PBSC-IL-3-pSTAT5 and CFU assays. Warming events were also simulated to emulate impaired potency products. RESULTS: Optimization led to minor protocol adjustments, such as removal of the red blood cell lysis step, the addition of a formaldehyde fixation step and an increase in anticoagulant concentration. The PBSC-IL-3-pSTAT5 assay discriminated between normal and impaired samples and identified 100% (18 of 18) of the impaired samples, thus showing better specificity than the CFU assay. CONCLUSIONS: The updated IL-3-pSTAT5 potency assay has several important advantages, such as accelerating the release of autologous stem cell products and enabling the detection of potentially impaired products. The assay could also be used to rapidly assess the potency of any cryopreserved allogeneic stem cell product, such as those processed during the coronavirus disease 2019 pandemic.

Cord blood beyond transplantation: can we use the experience to advance all cell therapies?

Unrelated cord blood (CB) units, already manufactured, fully tested and stored, are high-quality products for haematopoietic stem cell transplantation and cell therapies, as well as an optimal starting material for cell expansion, cell engineering or cell re-programming technologies. CB banks have been pioneers in the development and implementation of Current Good Manufacturing Practices for cell-therapy products. Sharing their technological and regulatory experience will help advance all cell therapies, CB-derived or not, particularly as they transition from autologous, individually manufactured products to stored, 'off-the shelf' treatments. Such strategies will allow broader patient access and wide product utilisation.

ASSESSMENT OF RADIOPROTECTIVE ACTION OF BASIDIOMYCOTIC MELANIN PIGMENTS ON THE HEMATOPOIETIC SYSTEM OF Balb/C MICE UNDER EXPOSURE TO IONIZING RADIATION IN SUBLETHAL DOSE. / OTsINKA RADIOPROTEKTORNOÏ DIÏ MELANINOVYKh PIGMENTIV BAZYDIOMITsETIV NA GEMOPOETYChNU SYSTEMU MYShEY̆ Balb/C PRY OPROMINENNI IONIZUIuChOIu RADIATsIIeIu U SUBLETAL'NIY̆ DOZI.

OBJECTIVE: Assessment of radioprotective action of basidiomycotic melanin pigments on hematopoietic stem and progenitor bone marrow cells of Balb/C mice in case of exposure to ionizing radiation in sublethal dose. MATERIALS AND METHODS: Using original method of cultivation in gel diffusion chambers in vivo of bone marrow cells of Balb/C mice we investigated the colony-forming efficiency of hematopoietic progenitor cells of the ani- mals, which were exposed to ionizing radiation action in sublethal dose, in case of treatment with melanin pig- ments solution of basidiomycotic fungi as radioprotector. RESULTS AND CONCLUSIONS: Investigation of functional activity of bone marrow progenitor cells of Balb/C mice allowed assessing their hematopoiesis state in case of ionizing radiation action, as well as in case of previous treat- ment of the animals with the solution of melanin pigments as radioprotector. It was determined that under the influence of ionizing radiation the colony-forming activity of mice bone marrow has decreased comparing to con- trol. Solution of melanin pigments was able to enhance the functional activity of bone marrow of irradiated ani- mals. Obtained results of radioprotective action of basidiomycotic melanin pigments solution on irradiated stem cells and their descendants (progenitor cells) may become the evidence for development of the protective means for human organism from the injuring action of ionizing radiation.

Further assessment of stochastic proliferation and its potential application to hematopoietic scaling across species.

Spleen colony-forming unit (CFU-s) growth in spleen colonies is a stochastic process in which CFU-s, with each cell division, can either self-renew or differentiate, but not both. The fundamental parameter governing this process is p, or the probability of CFU-s self-renewing. Previously, when CFU-s growth was modeled by Monte Carlo simulations, p was kept constant during the 20 cell cycles required for the modeling. However, it is known that CFU-s self-renewal undergoes decline with proliferation. In the present study, this was taken into consideration, such that p was forced to undergo a small decline with each cell division. These new Monte Carlo calculations give an improved fit to CFU-s cumulative growth curves as compared with those calculations using fixed p. This new model, referred to as the variable p model, offers an explanation as to how large mammals can amplify marrow output from stem cell compartments that are no larger than those found in small mammals. It is a model in which small changes in active stem cell aging generate disproportionally large increases in the size of active stem cell clones.

Clonal and Quantitative In Vivo Assessment of Hematopoietic Stem Cell Differentiation Reveals Strong Erythroid Potential of Multipotent Cells.

Hematopoiesis is arguably one of the best understood stem cell systems; however, significant challenges remain to reach a consensus understanding of the lineage potential, heterogeneity, and relationships of hematopoietic stem and progenitor cell populations. To gain new insights, we performed quantitative analyses of mature cell production from hematopoietic stem cells (HSCs) and multiple hematopoietic progenitor populations. Assessment of the absolute numbers of mature cell types produced by each progenitor cell revealed a striking erythroid dominance of all myeloid-competent progenitors assessed, accompanied by strong platelet reconstitution. All populations with myeloid potential also produced robust numbers of red blood cells and platelets in vivo. Clonal analysis by single-cell transplantation and by spleen colony assays revealed that a significant fraction of HSCs and multipotent progenitors have multilineage potential at the single-cell level. These new insights prompt an erythroid-focused model of hematopoietic differentiation.

Aldehyde dehydrogenase activity in cryopreserved cord blood cells for quality assessment prior to transplantation.

In umbilical cord blood transplantation (UCBT), the number of cluster of differentiation (CD)34+ cells and colony­forming units (CFUs) in the cord blood (CB) graft positively correlate with patient survival. Therefore, these parameters are currently used for quality assessment of the cryopreserved CB cells in the attached segment that is considered representative of the CB in the main bag prior to UCBT. Since aldehyde dehydrogenase (ALDH) activity is high in hematopoietic stem cells, the number of ALDH­bright (ALDHbr) cells was examined in comparison with the number of CD34+ cells and CFUs for the quality assessment of CB units. In the cryopreserved main bag, the number of ALDHbr cells in the CB unit exhibited positive correlation with the number of CD34+ cells, and with CFU­granulocytes/macrophages and total CFU counts. Furthermore, the concentration of ALDHbr cells in the cryopreserved attached segment was not significantly different compared to that of the main bag, suggesting that the attached segment is representative of the main bag. In conclusion, the present study suggested that ALDHbr cell counts in the cryopreserved attached segments may serve as a quality assessment indicator for CB units prior to UCBT.

Isolation and functional assessment of mouse skeletal stem cell lineage.

There are limited methods available to study skeletal stem, progenitor, and progeny cell activity in normal and diseased contexts. Most protocols for skeletal stem cell isolation are based on the extent to which cells adhere to plastic or whether they express a limited repertoire of surface markers. Here, we describe a flow cytometry-based approach that does not require in vitro selection and that uses eight surface markers to distinguish and isolate mouse skeletal stem cells (mSSCs); bone, cartilage, and stromal progenitors (mBCSPs); and five downstream differentiated subtypes, including chondroprogenitors, two types of osteoprogenitors, and two types of hematopoiesis-supportive stroma. We provide instructions for the optimal mechanical and chemical digestion of bone and bone marrow, as well as the subsequent flow-cytometry-activated cell sorting (FACS) gating schemes required to maximally yield viable skeletal-lineage cells. We also describe a methodology for renal subcapsular transplantation and in vitro colony-formation assays on the isolated mSSCs. The isolation of mSSCs can be completed in 9 h, with at least 1 h more required for transplantation. Experience with flow cytometry and mouse surgical procedures is recommended before attempting the protocol. Our system has wide applications and has already been used to study skeletal response to fracture, diabetes, and osteoarthritis, as well as hematopoietic stem cell-niche interactions in the bone marrow.

Investigating the Implications of a Variable RBE on Proton Dose Fractionation Across a Clinical Pencil Beam Scanned Spread-Out Bragg Peak.

PURPOSE: To investigate the clinical implications of a variable relative biological effectiveness (RBE) on proton dose fractionation. Using acute exposures, the current clinical adoption of a generic, constant cell killing RBE has been shown to underestimate the effect of the sharp increase in linear energy transfer (LET) in the distal regions of the spread-out Bragg peak (SOBP). However, experimental data for the impact of dose fractionation in such scenarios are still limited. METHODS AND MATERIALS: Human fibroblasts (AG01522) at 4 key depth positions on a clinical SOBP of maximum energy 219.65 MeV were subjected to various fractionation regimens with an interfraction period of 24 hours at Proton Therapy Center in Prague, Czech Republic. Cell killing RBE variations were measured using standard clonogenic assays and were further validated using Monte Carlo simulations and parameterized using a linear quadratic formalism. RESULTS: Significant variations in the cell killing RBE for fractionated exposures along the proton dose profile were observed. RBE increased sharply toward the distal position, corresponding to a reduction in cell sparing effectiveness of fractionated proton exposures at higher LET. The effect was more pronounced at smaller doses per fraction. Experimental survival fractions were adequately predicted using a linear quadratic formalism assuming full repair between fractions. Data were also used to validate a parameterized variable RBE model based on linear α parameter response with LET that showed considerable deviations from clinically predicted isoeffective fractionation regimens. CONCLUSIONS: The RBE-weighted absorbed dose calculated using the clinically adopted generic RBE of 1.1 significantly underestimates the biological effective dose from variable RBE, particularly in fractionation regimens with low doses per fraction. Coupled with an increase in effective range in fractionated exposures, our study provides an RBE dataset that can be used by the modeling community for the optimization of fractionated proton therapy.

Risk assessment of a cold argon plasma jet in respect to its mutagenicity.

Cold atmospheric pressure plasmas represent a favorable option for the treatment of heat sensitive materials and human or animal tissue. Beneficial effects have been documented in a variety of medical conditions, e.g., in the treatment of chronic wounds. It is assumed that the main mechanism of the plasma's efficacy is mediated by a stimulating dissipation of energy via radiation and/or chemical energy. Although no evidence on undesired side effects of a plasma treatment has yet been presented, skepticism toward the safety of the exposure to plasma is present. However, only little data regarding the mutagenic potential of this new treatment option is available. Accordingly, we investigated the mutagenic potential of an argon plasma jet (kinpen) using different testing systems in accordance with ISO norms and multiple cell lines: a HPRT1 mutation assay, a micronucleus formation assay, and a colony formation assay. Moderate plasma treatment up to 180 s did not increase genotoxicity in any assay or cell type investigated. We conclude that treatment with the argon plasma jet kinpen did not display a mutagenic potential under the test conditions applied and may from this perspective be regarded as safe for the use in biomedical applications.

Comparative Assessment of Oral Mesenchymal Stem Cells Isolated from Healthy and Diseased Tissues.

The aim of the present study was to isolate human mesenchymal stem cells (MSCs) from palatal connective and periodontal granulation tissues and to comparatively evaluate their properties. MSCs were isolated using the explant culture method. Adherence to plastic, specific antigen makeup, multipotent differentiation potential, functionality, and ultrastructural characteristics were investigated. The frequency of colony-forming unit fibroblasts for palatal-derived mesenchymal stem cells (pMSCs) was significantly higher than that of granulation tissue-derived mesenchymal stem cells (gtMSCs). A significantly higher population doubling time and lower migration potential were recorded for gtMSCs than for pMSCs. Both cell lines were positive for CD105, CD73, CD90, CD44, and CD49f, and negative for CD34, CD45, and HLA-DR, but the level of expression was different. MSCs from both sources were relatively uniform in their ultrastructure. Generally, both cell lines possessed a large, irregular-shaped euchromatic nucleus, and cytoplasm rich in mitochondria, lysosomes, and endoplasmic reticulum. The periphery of the plasma membrane displayed many small filopodia. MSCs from both cell lines were successfully differentiated into osteogenic, adiopogenic, and chondrogenic lineages. Both healthy and diseased tissues may be considered as valuable sources of MSCs for regenerative medicine owing to the high acceptance and fewer complications during harvesting.

Assessment of cell viability, early apoptosis, and hematopoietic potential in umbilical cord blood units after storage.

BACKGROUND: Successful hematopoietic stem cell transplantation using stored umbilical cord blood (CB) largely depends on cell dose and quality of CB units. The aim of this study was to assess the degree of early apoptosis, in addition to cell viability and hematopoietic potential, in umbilical CB units after storage. STUDY DESIGN AND METHODS: Sixty CB units that had been cryopreserved for up to 8 years in a single public CB bank were investigated. After the CB units were thawed, cell viability and early apoptosis of total nucleated cells (TNCs), mononuclear cells (MNCs), and CD34+ cells were determined using flow cytometric method based on 7-aminoactinomycin D (7-AAD) and annexin V staining. Next, clonogenic assays to predict graft potency were performed. RESULTS: Postthawing cell viability values determined by 7-AAD were as follows: TNCs, 78.8% ± 5.8%; MNCs, 88.4% ± 5.8%; and CD34+ cells, 94.1% ± 3.2%. Cell viability values using 7-AAD and annexin V dual staining were as follows: TNCs, 71.2% ± 11.3%; MNCs, 83.1% ± 7.0%; and CD34+ cells, 88.8% ± 6.0%. Early apoptotic cells (7-AAD-negative and annexin V-positive cells) in TNCs, MNCs, and CD34+ cells were 6.4% ± 3.5%, 5.4% ± 3.1%, and 5.3% ± 4.1%, respectively. The corrected colony-forming unit-granulocyte-macrophage content per 100 CD34+ cells was 67.5 ± 48.7. CONCLUSIONS: Postthawing cell viability determined by flow cytometric methods was in the following order: TNCs < MNCs < CD34+ cells. CD34+ cell viability was nearly identical to that of fresh CB 48 hours after collection. Necrosis or apoptosis in cryopreserved CB units did not accelerate during storage.

Stem cell-derived systems in toxicology assessment.

Industrial sectors perform toxicological assessments of their potential products to ensure human safety and to fulfill regulatory requirements. These assessments often involve animal testing, but ethical, cost, and time concerns, together with a ban on it in specific sectors, make appropriate in vitro systems indispensable in toxicology. In this study, we summarize the outcome of an EPAA (European Partnership of Alternatives to Animal Testing)-organized workshop on the use of stem cell-derived (SCD) systems in toxicology, with a focus on industrial applications. SCD systems, in particular, induced pluripotent stem cell-derived, provide physiological cell culture systems of easy access and amenable to a variety of assays. They also present the opportunity to apply the vast repository of existing nonclinical data for the understanding of in vitro to in vivo translation. SCD systems from several toxicologically relevant tissues exist; they generally recapitulate many aspects of physiology and respond to toxicological and pharmacological interventions. However, focused research is necessary to accelerate implementation of SCD systems in an industrial setting and subsequent use of such systems by regulatory authorities. Research is required into the phenotypic characterization of the systems, since methods and protocols for generating terminally differentiated SCD cells are still lacking. Organotypical 3D culture systems in bioreactors and microscale tissue engineering technologies should be fostered, as they promote and maintain differentiation and support coculture systems. They need further development and validation for their successful implementation in toxicity testing in industry. Analytical measures also need to be implemented to enable compound exposure and metabolism measurements for in vitro to in vivo extrapolation. The future of SCD toxicological tests will combine advanced cell culture technologies and biokinetic measurements to support regulatory and research applications. However, scientific and technical hurdles must be overcome before SCD in vitro methods undergo appropriate validation and become accepted in the regulatory arena.

Isolation and functional assessment of cutaneous stem cells.

The epidermis and associated appendages of the skin represent a multi-lineage tissue that is maintained by perpetual rounds of renewal. During homeostasis, turnover of epidermal lineages is achieved by input from regionalized keratinocytes stem or progenitor populations with little overlap from neighboring niches. Over the last decade, molecular markers selectively expressed by a number of these stem or progenitor pools have been identified, allowing for the isolation and functional assessment of stem cells and genetic lineage tracing analysis within intact skin. These advancements have led to many fundamental observations about epidermal stem cell function such as the identification of their progeny, their role in maintenance of skin homeostasis, or their contribution to wound healing. In this chapter, we provide a methodology to identify and isolate epidermal stem cells and to assess their functional role in their respective niche. Furthermore, recent evidence has shown that the microenvironment also plays a crucial role in stem cell function. Indeed, epidermal cells are under the influence of surrounding fibroblasts, adipocytes, and sensory neurons that provide extrinsic signals and mechanical cues to the niche and contribute to skin morphogenesis and homeostasis. A better understanding of these microenvironmental cues will help engineer in vitro experimental models with more relevance to in vivo skin biology. New approaches to address and study these environmental cues in vitro will also be addressed.

Characteristics of stem cells derived from rat fascia: in vitro proliferative and multilineage potential assessment.

Fascia­derived stem cells (FDSCs) were previously isolated from the fascia of the gluteus maximus of the rat. However, the use of FDSCs as a cell source for musculoskeletal tissue engineering has not been compared with that of adipose­derived stem cells (ADSCs) and bone marrow­derived mesenchymal stem cells (BMSCs). Therefore, the present study aimed to compare the mesenchymal stem cell (MSC) and self­renewal stem cell markers, proliferative capacity and multilineage differentiation potential of these stem cells in vitro. The MSC and embryonic stem cell (ESC) marker profiles were compared using flow cytometry and quantitative polymerase chain reaction (qPCR). Their proliferative capacities were compared using 5­bromo­2'­deoxyuridine and MTT assays. Their osteogenic, adipogenic and chondrogenic differentiation potentials were compared using standard staining assays and qPCR. The FDSCs possessed similar cell morphology and immunophenotypic profiles with BMSCs and ADSCs. FDSCs demonstrated a similar expression pattern of ESC markers with ADSCs, which has higher expression of sex determining region Y­box (Sox)2 and octamer­binding transcription factor 4, and lower expression of Krüppel­like factor 4, when compared with BMSCs. FDSCs exhibited higher proliferation under serum­deprived conditions (0.5% FBS growth medium), and attained higher expression levels of collagen type I, α 2 and type II, α 1 as well as Sox9 mRNA than ADSCs and BMSCs upon chondrogenic induction. An increased amount of proteoglycan deposition was also observed in the FDSC group. However, lower levels of adipogenic and osteogenic marker expression in FDSCs were detected compared with ADSCs and BMSCs upon adipogenic and osteogenic induction, respectively. FDSCs possessed high chondrogenic potential, low osteogenic and adipogenic differentiation potential and were responsive to the induction signals for collagen­rich fascial structure regeneration. Therefore, FDSCs may represent an improved alternative cell source to conventional ADSCs and BMSCs for musculoskeletal tissue repair and tissue engineering, particularly for collagen­rich structures with poor vasculature.

Stochastic modeling of stress erythropoiesis using a two-type age-dependent branching process with immigration.

The erythroid lineage is a particularly sensitive target of radiation injury. We model the dynamics of immature (BFU-E) and mature (CFU-E) erythroid progenitors, which have markedly different kinetics of recovery, following sublethal total body irradiation using a two-type reducible age-dependent branching process with immigration. Properties of the expectation and variance of the frequencies of both types of progenitors are presented. Their explicit expressions are derived when the process is Markovian, and their asymptotic behavior is identified in the age-dependent (non-Markovian) case. Analysis of experimental data on the kinetics of BFU-E and CFU-E reveals that the probability of self-renewal increases transiently for both cell types following sublethal irradiation. In addition, the probability of self-renewal increased more for CFU-E than for BFU-E. The strategy adopted by the erythroid lineage ensures replenishment of the BFU-E compartment while optimizing the rate of CFU-E recovery. Finally, our analysis also indicates that radiation exposure causes a delay in BFU-E recovery consistent with injury to the hematopoietic stem/progenitor cell compartment that give rise to BFU-E. Erythroid progenitor self-renewal is thus an integral component of the recovery of the erythron in response to stress.

Functional assessment of hematopoietic niche cells derived from human embryonic stem cells.

To evaluate hematopoietic niche cell populations isolated from human embryonic stem cells (hESCs), we tested the ability of hESC-derived stromal lines to support CD34(+) umbilical cord blood (UCB)- and hESC-derived CD34(+)45(+) cells in long-term culture initiating cell (LTC-IC) assays. Specifically, these hematopoietic populations were cocultured with hESC-derived mesenchymal stromal cells (hESC-MSCs) and hESC-derived endothelial cells (hESC-ECs), and then assessed for their LTC-IC potential in comparison to coculture with bone marrow (BM)-derived MSCs and the mouse stromal line M2-10B4. We found that the hESC-derived stromal lines supported LTC-ICs from UCB similar to M2-10B4 cells and better than BM-MSCs. However, none of the stromal populations supported LTC-IC from hESC-derived CD34(+)45(+) cells. Engraftment data using the output from LTC-IC assays showed long-term repopulation (12 weeks) of NSG mice to correlate with LTC-IC support on a given stromal layer. Therefore, hESC-derived stromal lines can be used to efficiently evaluate putative hematopoietic stem/progenitor cells derived from hESCs or other cell sources.

Combining the LKB NTCP model with radiosensitivity parameters to characterize toxicity of radionuclides based on a multiclonogen kidney model: a theoretical assessment.

The Lyman-Kutcher-Burman (LKB) normal tissue complication probability (NTCP) model is often used to estimate the damage level to normal tissue. However, it does not manifestly involve the influence of radiosensitivity parameters. This work replaces the generalized mean equivalent uniform dose (gEUD) with the equivalent uniform dose (EUD) in the LKB model to investigate the effect of a variety of radiobiological parameters on the NTCP to characterize the toxicity of five types of radionuclides. The dose for 50 % complication probability (D (50)) is replaced by the corresponding EUD for 50 % complication probability (EUD(50)). The properties of a variety of radiobiological characteristics, such as biologically effective dose (BED), NTCP, and EUD, for five types of radioisotope ((131)I, (186)Re, (188)Re, (90)Y, and (67)Cu) are investigated by various radiosensitivity parameters such as intrinsic radiosensitivity α, alpha-beta ratio α/ß, cell repair half-time, cell mean clonogen doubling time, etc. The high-energy beta emitters ((90)Y and (188)Re) have high initial dose rate and mean absorbed dose per injected activity in kidney, and their kidney toxicity should be of greater concern if they are excreted through kidneys. The radiobiological effect of (188)Re changes most sharply with the radiobiological parameters due to its high-energy electrons and very short physical half-life. The dose for a probability of 50% injury within 5y (D (50/5)) 28 Gy for whole-kidney irradiation should be adjusted according to different radionuclides and different radiosensitivity of individuals. The D (50/5) of individuals with low α/ß or low α, or low biological clearance half-time, will be less than 28 Gy. The 50 % complication probability dose for (67)Cu and (188)Re could be 25 Gy and 22 Gy. The same mean absorbed dose generally corresponds to different degrees of damage for tissues of different radiosensitivity and different radionuclides. The influence of various radiobiological parameters should be taken into consideration in the NTCP model.

Geant4-Simulations for cellular dosimetry in nuclear medicine.

The application of unsealed radionuclides in radiobiological experiments can lead to intracellular radionuclide uptake and an increased absorbed dose. Accurate dose quantification is essential to assess observed radiobiological effects. Due to small cellular dimensions direct dose measurement is impossible. We will demonstrate the application of Monte Carlo simulations for dose calculation. Dose calculations were performed using the Geant4 Monte Carlo toolkit, wherefore typical experimental situations were designed. Dose distributions inside wells were simulated for different radionuclides. S values were simulated for spherical cells and cell monolayers of different diameter. Concomitantly experiments were performed using the PC Cl3 cell line with mediated radionuclide uptake. For various activity distributions cellular survival was measured. We yielded S values for dose distribution inside the wells. Calculated S values for a single cell are in good agreement to S values provided in the literature (ratio 0.87 to 1.07). Cross-dose is up to ten times higher for Y-90. Concomitantly performed cellular experiments confirm the dose calculation. Furthermore the necessity of correct dose calculation was shown for assessment of radiobiological effects after application of unsealed radionuclides. Thereby the feasibility of using Geant4 was demonstrated.

Interlaboratory assessment of a novel colony-forming unit assay: a multicenter study by the cellular team of Biomedical Excellence for Safer Transfusion (BEST) collaborative.

BACKGROUND: Interlaboratory scoring performances were determined using a traditional 14-day colony-forming unit (CFU) assay and a new 7-day CFU assay. STUDY DESIGN AND METHODS: Digital images of colonies were utilized to train personnel at each site. A central laboratory inoculated methylcellulose with progenitors and sent the samples by overnight courier to participating labs for plating. RESULTS: Colony counts from two digital images showed greater variability by novice counters (coefficients of variation [CV], 18.5 and 23.0%; n = 8) than for experienced staff (CV, 7.3 and 4.8%; n = 5). CFU assays plated immediately, 24 and 48 hours after methylcellulose inoculation displayed 39.5 CFU, 37.1 ± 10.6 (CV, 28%) and 34.8 ± 8.5 (CV, 24%) colonies for the 7-day assay and 39.5 CFU, 39.1 ± 9.9 (CV, 25%) and 37.1 ± 10.6 (CV, 28%) colonies for the 14-day assay, respectively. Overall, no significant differences in colony counts were noted between assays (p = 0.68). Also, no differences in CFU counts were seen when assays were set up immediately, 24 and 48 hours after methylcellulose inoculation (14-day p = 0.695; 7-day p = 0.632). CONCLUSION: Total CFUs obtained in 7- and 14-day CFU assays are comparable and show similar levels of interlaboratory variability. The major source of this variability is due to differences in how CFU plates are scored by individuals at different sites. UCB progenitor cells can be maintained in methylcellulose-based media at room temperature for up to 48 hours prior to transport without a significant loss in CFUs.

Myocardial implantation of a combination stem cell product by using a transendocardial MYOSTAR injection catheter: A technical assessment.

AIM: Different types of progenitor cells have been used to improve cardiac conditions after myocardial infarction (MI). Results have shown that while the infusion of a single cell type is safe and feasible, efficacy is modest. Recently, the use of a combination, rather than a single, stem cell product has emerged as an attractive option to improve cardiac outcome after a MI. Before initiating a phase II clinical trial to assess safety and efficacy after the transendocardial infusion of a combination stem cell product, a bench testing assay was designed to validate that delivery through the injection catheter is not associated with cell loss/damage. The latter is important since mesenchymal stem cells (MSC), a component of the cell product, consist of large cells expressing matrix molecules and adhesive receptors. METHODS: The cell product (a mixture of mononuclear cells and MSC) was sequentially injected through a Myostar injection catheter. Exiting fractions were assessed for cell number, viability, capability to restart cell growth and immunophenotype. RESULTS: Cell recovery and viability were high. In turn, exiting cells preserved their biological properties and immunophenotype. CONCLUSIONS: Delivery of cells through a Myostar catheter is safe and not associated with changes in cell survival and/or properties.