Inventors : BULAVIN DMITRY [FR]; GRIGORASH BOGDAN [FR]
SUMMARY OF THE INVENTION
In the experimental part which follows, the inventors, using genetic, chemical and mechanical approaches to manipulate senescent cells, showed that removal of p16Hi9h cells results in the 4 factor-induced reprogramming into embryonic stem cells with totipotent potential. Established induced stem cells with totipotent potential (iTLS for induced totipotent-like stem cells) carried the markers of both pluripotency and the 2-Cell embryonic state and were readily forming blastocyst-like structures, blastoids, in vitro under defined chemical conditions. In vivo, formed blastoids were capable of implantation and embryonic development up to at least 7,5 days post coitum (dpc). They also identified senescence-dependent regulation of Nicotinamide N-methyltransferase (NNMT) as a key mechanism controlling S-adenosyl methionine (SAM) levels that required for regulation of global histone methylation, expression of 2C state genes including retroelements and acquisition of a totipotent potential. Here is provided the framework for generation of embryo-competent mouse iTLS, a procedure that could be applicable for other nonhuman species as well as human.
According to a first aspect, the present invention thus pertains to a method of preparing induced totipotent-like stem (iTLS) cells from a somatic cell population, said method comprising removing p16Hi9h senescent cells and/or preventing the appearance of p16Hi9h senescent cells from said cell population during reprogramming of said somatic cell population. The invention also relates to a method of obtaining a blastoid, by incubating an iTLS cell obtained according to the invention in appropriate conditions during 4 to 6 days. The resulting blastoid is also part of the present invention, and has the remarkable property of being able to implant and develop in vivo during at least 5 days in a pseudopregnant recipient animal.
Another object of the present invention is a method of obtaining a blastoid of at least 7.5 dpc from a somatic cell, by implanting a blastoid obtained in vitro according to the invention into the uterus of a pseudopregnant non-human animal, and removing the obtained blastoid from the animal at least 5 days later.
The resulting blastoid is also part of the present invention, and can advantageously be used as a cell material for regenerative medicine.