Development of the clinical grade protocols of placental HSCs cryopreservation, isolation and expansion for hematopoietic recovery

Cord blood hematopoietic stem cells are widely used for hematopoietic recovery in patients after intensive chemo- and radiotherapy. Low amount of HSCs in cord blood unit (CBU) is the great obstacle that prevents wide usage of CBU transplantation for treatment in adults. Term placenta contains significant amount of HSCs in vessel space as well as in extra vessel stromal tissue. It was found that myelopoiesis occurs in the placenta, leading to the formation of monocytes and erythroblasts during the first and second trimesters of human pregnancy. However, it remains unclear how the placental HSC renewal and differentiation occurs, which signaling pathways are involved in maintaining the dormancy, triggering placental proliferation, maintaining stemness and differentiating. The study of these key moments is crucial for the development of a protocol for the optimal isolation and expansion of human placental HSCs.

First, this research is focused on the study of biological features of placental HSCs in situ (transcriptome profile, signaling, self-renewal and differentiation regulations, niche properties), and the development of ex vivo condition for their expansion. The next step will be addressed to the clinical-grade protocols of placental HSCs isolation, expansion ex vivo and cryopreservation. We have already compared the HSCs recovery from placental tissues cryopreserved with different cryoprotectant composition (unpublished data).

 

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We developed the method of cryopreservation of placental tissue fragments and isolation of viable HSCs from frozen tissue that gives rise to colonies in vitro after 14 days of culturing in the MethoCult medium: (a) BFU-E, (b) CFU-GM, (c) CFU-M, and (d) CFU-GEMM (×50)

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Placental hematopoietic cells from both native and frozen full-term placental tissue consist of different subpopulations as CD34+/lowCD45hi (1), CD34++CD45hi (2), CD34+/lowCD45low/- (3), CD34++CD45low/- (4) and CD34+++CD45low/- (5)

In summary, as a main result of this research project we assumed the development of clinical grade SOPs for processing cryopreserved human term placental tissue unit as well as HSCs units expanded  ex vivo for hematopoietic recovery. The influence of cryopreservation on molecular and functional properties of HSCs will be revealed, and the mechanism of cryoresistance and nature of cryoinjuries of regional stem cells during complex tissue cryopreservation will be conducted.

 

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