Generation of mesenchymal stromal cells from cord blood: evaluation of in vitro quality parameters prior to clinical use

Abstract Abstract 4811 The number of hematopoietic stem and progenitor cells (HPCs) in cord blood units are limited and this can result in delayed engraftment. In vitro expansion of HPCs provides a perspective to overcome these limitations. Different combinations of cytokines as well as mesenchymal stromal cells (MSC) have been shown to separately support HPCs ex vivo expansion, but the combining effects are under evaluation. Data derived from ex vivo co-culture systems using MSC as a feeder layer suggest that cellular contacts could have a significant impact on expansion. We have evaluated the expansion rate of thawed cord blood samples (n=6) in a medium containing SCF (100 ng/ml) and G-CSF (100 ng/ml) plated over a pre-established bone marrow derived MSC layer in comparison to the absence of either MSC layer or cytokines. After 7 days cultures were demi-depopulated. At 14 days of culture adherent and non-adherent cells were harvested, counted and evaluated for antigens expression and clonogenic capacity. Immunophenotypic analysis was performed using CD34-PE, CD38-FITC, CD45-PE-Cy7, CD133-APC. Clonogenic assay was performed in semisolid methylcellulose culture medium (MethoCult, Stem Cell Technologies), CFU frequencies and total CFU numbers per cord blood sample were determined. After 14 days of culture, in the presence of MSC layer, an 11.2-fold increase (range 4.4–18.4) in total number of cells was observed, in comparison to a 4.8-fold increase (range1.1-10.35) in the absence of MSC layer. The presence of MSC layer generated a 4.3-fold increase (range 1.5–7.2) in the number of CD34 positive cells, compared to a 3.3-fold increase (range 0.9–5.7) in the absence of MSC; when considering the more immature CD34+/CD38− subpopulation the corresponding increase were 26.9-fold vs 2.85-fold, respectively. Moreover, the percentage of the CD34+/CD38− subpopulation was higher in the adherent compared to the non-adherent fraction (76% vs 15%). The selection effect given by the MSC layer was confirmed by the presence of hematopoiesis foci growing onto the MSC layer. Our data show that cord blood HPCs can be expanded in vitro, moreover the co-culture on a MSC layer shows a synergistic effect on TCN, CD34+ cells and on more primitive CD34+/CD38− cells. Therefore, a clinical protocol of cord blood HPCs and MSC co-culture could represent a promising approach for improving engraftment kinetics in cord blood transplant recipients. Disclosures: No relevant conflicts of interest to declare.

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Wound-healing potential of human umbilical cord blood–derived mesenchymal stromal cells in vitro—a pilot study

Cytotherapy ◽

10.1016/j.jcyt.2015.06.011 ◽

2015 ◽

Vol 17 (11) ◽

pp. 1506-1513 ◽

Cited By ~ 14

Author(s):

Hi-Jin You ◽

Sik Namgoong ◽

Seung-Kyu Han ◽

Seong-Ho Jeong ◽

Eun-Sang Dhong ◽

...

Keyword(s):

Wound Healing ◽

Pilot Study ◽

Cord Blood ◽

Umbilical Cord ◽

Mesenchymal Stromal Cells ◽

Umbilical Cord Blood ◽

Stromal Cells ◽

Human Umbilical Cord Blood ◽

Human Umbilical Cord

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Chondrogenic potential of mesenchymal stromal cells derived from equine bone marrow and umbilical cord blood

Veterinary and Comparative Orthopaedics and Traumatology ◽

10.3415/vcot-08-10-0107 ◽

2009 ◽

Vol 22 (05) ◽

pp. 363-370 ◽

Cited By ~ 52

Author(s):

T. G. Koch ◽

T. Heerkens ◽

K. Besonov ◽

P. D. Thomsen ◽

D. H. Betts ◽

...

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Umbilical Cord ◽

Mesenchymal Stromal Cells ◽

Umbilical Cord Blood ◽

Stromal Cells ◽

Supplementary Information ◽

Multiple Sources ◽

Chondrogenic Potential

Summary Objective: Orthopaedic injury is the most common cause of lost training days or premature retirement in the equine athlete. Cell-based therapies are a potential new treatment option in musculo-skeletal diseases. Mesenchymal stromal cells (MSC) have been derived from multiple sources in the horse including bone marrow and umbilical cord blood. The objective of this study was to provide an in vitro comparison of the chondrogenic potential in MSC derived from adult bone marrow (BM-MSC) and umbilical cord blood (CB-MSC). Results: MSC from both sources produced tissue with cartilage-like morphology that stained positive for proteoglycans and expressed cartilage markers. The CB-MSC pellets were larger and showed hyaline-like cartilage morphology as early as day six. Gene expression of collagen type 21, aggrecan and CD-RAP was higher in CB- than BM-MSC pel-lets. Expression of Sox9 mRNA was similar between CB- and BM-MSC pellets. Protein concentration of cartilage-derived retinoic acid sensitive protein was higher in culture medium from CB- than BM-MSC pellets. Conclusion: CB-MSC and BM-MSC were both capable of producing hyaline-like cartilage in vitro. However, in this study the MSC from umbilical cord blood appeared to have more chondrogenic potential than the BMMSC based on the cells tested and parameters measured.Supplementary Information for this paper is available on the VCOT website at www.VCOTonline.com.

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Equine Mesenchymal Stem/Stromal Cells Freeze-Dried Secretome (Lyosecretome) for the Treatment of Musculoskeletal Diseases: Production Process Validation and Batch Release Test for Clinical Use

Pharmaceuticals ◽

10.3390/ph14060553 ◽

2021 ◽

Vol 14 (6) ◽

pp. 553

Author(s):

Michela Mocchi ◽

Stefano Grolli ◽

Silvia Dotti ◽

Dario Di Silvestre ◽

Riccardo Villa ◽

...

Keyword(s):

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Musculoskeletal Diseases ◽

Biological Effects ◽

Critical Parameters ◽

Clinical Use ◽

Pharmaceutical Dosage Forms ◽

Batch Release ◽

Freeze Dried

In the last decades, it has been demonstrated that the regenerative therapeutic efficacy of mesenchymal stromal cells is primarily due to the secretion of soluble factors and extracellular vesicles, collectively known as secretome. In this context, our work described the preparation and characterization of a freeze-dried secretome (Lyosecretome) from adipose tissue-derived mesenchymal stromal cells for the therapy of equine musculoskeletal disorder. An intraarticular injectable pharmaceutical powder has been formulated, and the technological process has been validated in an authorized facility for veterinary clinical-use medicinal production. Critical parameters for quality control and batch release have been identified regarding (i) physicochemical properties; (ii) extracellular vesicle morphology, size distribution, and surface biomarker; (iii) protein and lipid content; (iv) requirements for injectable pharmaceutical dosage forms such as sterility, bacterial endotoxin, and Mycoplasma; and (v) in vitro potency tests, as anti-elastase activity and proliferative activity on musculoskeletal cell lines (tenocytes and chondrocytes) and mesenchymal stromal cells. Finally, proteins putatively responsible for the biological effects have been identified by Lyosecretome proteomic investigation: IL10RA, MXRA5, RARRES2, and ANXA1 modulate the inflammatory process RARRES2, NOD1, SERPINE1, and SERPINB9 with antibacterial activity. The work provides a proof-of-concept for the manufacturing of clinical-grade equine freeze-dried secretome, and prototypes are now available for safety and efficacy clinical trials in the treatment of equine musculoskeletal diseases

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