Strategy for the Generation of Engineered Bone Constructs Based on Umbilical Cord Mesenchymal Stromal Cells Expanded with Human Platelet Lysate

Abstract

Umbilical cord mesenchymal stromal cells (UC-MSC) are promising candidates for cell therapy due to their potent multilineage differentiation, enhanced self-renewal capacity, and immediate availability for clinical use. Clinical experience has demonstrated satisfactory biosafety profiles and feasibility of UC-MSC application in the allogeneic setting. However, the use of UC-MSC for bone regeneration has not been fully established. A major challenge in the generation of successful therapeutic strategies for bone engineering lies on the combination of highly functional proosteogenic MSC populations and bioactive matrix scaffolds. To address that, in this study we proposed a new approach for the generation of bone-like constructs based on UC-MSC expanded in human platelet lysate (hPL) and evaluated its potential to induce bone structures in vivo. In order to obtain UC-MSC for potential clinical use, we first assessed parameters such as the isolation method, growth supplementation, microbiological monitoring, and cryopreservation and performed full characterization of the cell product including phenotype, growth performance, tree-lineage differentiation, and gene expression. Finally, we evaluated bone-like constructs based on the combination of stimulated UC-MSC and collagen microbeads for in vivo bone formation. UC-MSC were successfully cultured from 100% of processed UC donors, and efficient cell derivation was observed at day 14 ± 3 by the explant method. UC-MSC maintained mesenchymal cell morphology, phenotype, high cell growth performance, and probed multipotent differentiation capacity. No striking variations between donors were recorded. As expected, UC-MSC showed tree-lineage differentiation and gene expression profiles similar to bone marrow- and adipose-derived MSC. Importantly, upon osteogenic and endothelial induction, UC-MSC displayed strong proangiogenic and bone formation features. The combination of hPL-expanded MSC and collagen microbeads led to bone/vessel formation following implantation into an immune competent mouse model. Collectively, we developed a high-performance UC-MSC-based cell manufacturing bioprocess that fulfills the requirements for human application and triggers the potency and effectivity of cell-engineered scaffolds for bone regeneration.

Figure 1

Initial characterization of UC-MSC according to three isolation methodologies. (a) Percentage of positive umbilical cords (UC) processed by each isolation method (n = 10‐12 donors per group). (b) Time of cell derivation (P0 to P1) for every isolation method (n = 10‐12 donors per group). (c) Population doubling time measured in early cell passages (P2 to P6) per isolation method (n = 10 donors per group). (d) Flow cytometry analyses of MSC identity markers (CD90, CD73, CD105, HLA-AB, HLA-DR, CD45, and CD34) as shown in total frequency. Level of expression for CD90, CD73, CD105, and HLA-Ab is presented as Median Fluorescence Intensity (MFI) (n = 4 donors per group). ∗ indicates p < 0.05, as evaluated by ANOVA.

Figure 2

Microbiological monitoring of umbilical cord tissue, transport and washing solutions, and culture media used during isolation and initial culture of UC-MSC. (a) Percentage of contaminated samples comparing cesarean and vaginal deliveries (n = 11 donors per group). (b) Microorganisms identified in contaminated donors (filled squares) collected in cesarean and vaginal deliveries. ∗ indicates p values < 0.05 when comparing cesarean vs. vaginal delivery as evaluated by Chi-squared and Fisher exact tests.

Figure 3

Impact of the use of human platelet lysate (hPL) as a medium supplement for cell culture of UC-MSC. (a) Proliferation kinetics of UC-MSC as measured by population doubling time (hours), population doubling levels (PDL), and cumulative population doublings (CPD) per passage. Pooled batches of hPL from different blood groups were evaluated and compared with Fetal Bovine Serum (FBS) supplement (n = 4 donors per group). (b) Comparison of human cytokine levels (pg/mL) measured in different hPL batches obtained from blood donors (n = 3 per blood group). ∗ indicates p < 0.05 when comparing FBS vs. hPL as evaluated by ANOVA and Tukey's multiple comparison. § indicates p < 0.05 as tested by ANOVA.

Figure 4

Viability, recovery, and growth rate of UC-MSC evaluated after cryopreservation. (a) Cell viability after thawing represented as percentage of alive cells obtained (n = 7 donors per group). (b) Cell recovery after thawing (n = 7 donors per group). (c) Population doubling levels (PDL) and (d) cell doubling time assessed in UC-MSC cultures (n = 12 per group) obtained after different cryopreservation methods.

Figure 5

Long-term proliferation kinetics and potential cell yield of UC-MSC cultures. (a) Population doubling levels (PDL) and (b) cumulative population doublings (CPD) of UC-MSC cultures from four different umbilical cord (UC) donors maintained for up to 23 passages. (c) Theoretical total cell counts calculated according to cell growth kinetics observed in the evaluated UC donors.

Figure 6

Mesenchymal lineage differentiation of UC-MSC. (a) Cultured UC-MSC (passages 5-6) were induced towards osteogenic and adipogenic lineages, and differentiation was verified by Alizarin Red and Oil Red staining, respectively. For chondrogenic differentiation, UC-MSC (n = 3 donors) pellets were incubated in chondrogenic induction media and micromasses were evaluated by Masson's trichrome staining. Bone marrow- (BM-) and adipose tissue- (AD-) derived MSC were used as controls for differentiation. Embedded images show representative size of cartilage-like pellets (bar indicates 100 μm). Relative gene expression (qRT-PCR analysis) of adipocyte-, osteocyte-, and chondrocyte-related genes in UC-MSC (n = 3 donors) after 14 and 21 days of differentiation. Gene expression levels of (b) SPP1, (c) BGLAP, (d) FABP4, (e) PPARγ, (f) COMP and (g) FMOD are presented as fold increase relative to the housekeeping gene. Gene expression was also evaluated in BM and AD-derived MSC and used as controls.

Figure 7

In vivo formation of bone-like scaffolds based on UC-MSC. (a) Expression of endothelial and MSC markers in cells treated with endothelial induction (EM) or growth media. (b) Concentration of growth and angiogenic factors in EM, OM-treated UC-MSC, or nontreated (basal) controls. (c) Bone-like tissue formation at 12 weeks after transplantation of UC-MSC-microbeads in mice. (d) Mineralization zones are evident at the upper part (circles) and angiogenesis (arrows) in bone-like tissue formed from UC-MSC. (e) Hematoxylin and eosin staining evidence the presence of osteoid cells of immature appearance (dotted line) in bone-like tissue formed from UC-MSC scaffolds. (f) Cell migration outside explants of in vivo-formed bone-like tissue after 48 h of culture. Representative micrographs of bone constructs in growth medium (g) and differentiation medium (h) stained for Alizarin Red after 14 days of culture. Representative micrographs of bone constructs without (i) and with UC-MSC (j) stained with Masson's trichrome cultured for 14 days. Micrographs of hematoxylin and eosin (k) and collagen (l) staining of 14-day cultured scaffolds containing UC-MSC. Differences between expressions of different markers were compared. ∗ indicates p < 0.05. Bars indicate 100 μm.