ISSN 1866-8836
Клеточная терапия и трансплантация

DT-05. Influence of placenta-derived mesenchymal stromal cell on clonogenic potential of deposited cord blood hematopoietic stem cells

Maria A. Novikova, Yanina I. Isaikina, Yuliya V. Savich, Elena G. Liakh

Belarusian Research Center for Pediatric Oncology, Hematology and Immunology, Minsk, Republic of Belarus


Contact: Maria A. Novikova, phone: +375 (29) 788-94-21, e-mail: mariya_novikova32mail.ru

doi 10.18620/ctt-1866-8836-2023-12-3-1-176

Summary

Due to the role of placenta as a transient organ of the fetal hematopoiesis, the placenta-derived mesenchymal stem cells (MSCs) have a supportive hematopoietic capacity. Of particular interest are MSC derived from different anatomic placental tissues, with respect to their functional characteristics. Umbilical cord blood (UCB) is a proven source of hematopoietic stem cells (HSC). However, low volumes and numbers of HSC obtained from a single CB unit limit their application in adult patients. Supportive capacity of MSCs in hematopoiesis could represent an interest for their usage as a feeder layer (FL) to UCB HSC expansion in vitro. The aim of our study was to compare the influence of MSCs derived from different anatomic areas of placenta and from bone marrow upon the UCB HSC colony forming ability.

Materials and methods

MSCs were isolated from decidua, chorionic villi, amniotic membrane from full-term placentas of 5 healthy donors and from bone marrow (BM) of 4 donors then being expanded in vitro. MSC cell populations were analyzed by flow cytometry and characterized by a high expression of typical mesenchymal cell markers, i.e., CD90, CD105, CD73. UCB units (n=5) were frozen in the cryopreserving medium containing 10% dimethylsulfoxide and stored at -196ºС for 24-29 months. MNCs from UCB were thawed and plated at a density 1×106 cell/well over irradiated (30Gy) MSC feeder layer from placenta, or from BM MSCs at a ratio of 10:1 in RPMI medium supplemented with 15% fetal bovine serum and growth factors (SCF, TPO, FLt-3l, IL-6) for 7 days. Proliferative ability of hematopoietic progenitor cells was assessed by the colony-forming unit (CFU) assay. Expanded and non-expanded cells were cultured for 14 days in methylcellulose medium (StemCell Technologies, Canada) at 37°C, 5% CO2 and 90% humidity. The resulting colonies have been counted using a bright-field microscope and classified into CFU-G, CFU-M, CFU-GM, BFU-E, or CFU-Mix.

Results

The CFU assays of UCB HSC after the 7-day expansion, regardless of the MSC source, showed 1.8-fold increase of the total CFU numbers in the co-culture systems compared to day 0 (р<0.05). The cultures without MSC feeders seemed to show 1.1-fold increase in the total number of CFUs. MSCs from either source promoted proliferation of granulocyte and macrophage precursors. The initial number of CFU-GM 16 (4 to 20) was increased after co-culture with MSCs from decidual tissue up to 64 (40-72); from chorionic villi, up to 44 (10 to 112); from amniotic membrane, up to72 (36-88), and with BM MSCs, up to 36 (20 to 72) (p<0.05). Of note, both BM MSCs and MSCs from placenta were able to support the maintenance of most primitive myelopoietic progenitors: the number of CFU-GEMM before expansion was 6 (4-12), then being equal to 8 (0-16) after cultivation with BM MSCs, and 5 (0-16) with placental MSCs. A shift in the type of colonies produced was observed, as follows: on day 0 the percentage of CFU-GM was 71%; BFU-E, 25%; CFU-GEMM, 4%, whereas the BFU-Es rate after cultivation with MSCs from decidual tissue was reduced to 12%; with MSCs from amniotic membrane, 11%. Accordingly, the percentage of CFU-GM colonies showed a significant increase (р<0.05).

Conclusion

MSCs derived from different placental tissues and BM MSC could be applied for in vitro expansion of UCB HSCs stored at ultralow temperatures, and provide increased numbers of bipotent progenitor cells (CFU-GM), as well as to retain the levels of early multipotent myelopoiesis precursor cells (CFU-GEMM).

Keywords

Mesenchymal stem cells, umbilical cord blood, placenta, colony-forming unit.


Supplement 12-3
09/30/2023

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doi 10.18620/ctt-1866-8836-2023-12-3-1-176

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