In vitro razvoj i regeneracija primarnih staničnih kultura korteksa oposuma Monodelphis domestica

Autor: Mogorović, Sara
Přispěvatelé: Ban, Jelena, Mladinić Pejatović, Miranda, Bradshaw, Nicholas James
Jazyk: angličtina
Rok vydání: 2022
Popis: Central nervous system (CNS) sustained injuries in mammals often result in severe and irreversible damage, with CNS regeneration being limited due to the restricted regenerative potential of neurons. Yet, CNS regeneration is possible in embryos, and its regenerative potential stops very early after birth. Monodelphis domestica, the grey short-tailed opossum, is a pouch-less marsupial born at a very immature state, equivalent to embryonal day (E)13 in rats or E12.5 in mice. The initial development of their cortical plate occurs at postnatal day (P)3-5 and gliogenesis begins around P18. Moreover, these opossums have an extraordinary ability to fully regenerate their spinal cord up to two weeks after birth, with their regenerative ability declining with the rise of non-neuronal cells. Therefore, they are a great animal model to study CNS development and regeneration potential in vitro. Primary cell cultures derived from their cortices were obtained and examined at different developmental ages, P6 and P17. The cellular composition of primary cortical cell cultures was best characterized in P6 opossums with immunofluorescence microscopy. TUJ1+ neuronal population rose from 60% at days in vitro (DIV)1 to its peak 84% of neurons at DIV5, and further declined to 44% at DIV13. Astrocytes showed maturation from DIV5 to DIV13, accounting for 28% to 32% of GFAP+ cells, respectively. Neuronal and non-neuronal cells’ regenerative potential within the CNS was examined with an induced injury in both P6 and P17 cortical cultures, performed with a scratch assay. P6 cultures did show a better regenerative performance, as neurons regenerated and grew their axons across the cut site, forming growth cones (GCs) which differed in size with age. Astrocytes polarized their processes along the cut site or migrated to the cut, resembling glial scar formation. Opossum primary cortical cultures provide, therefore, an additional and novel source of mammalian cells for in vitro investigations.
Ozljede središnjeg živčanog sustava (SŽS) kod sisavaca često rezultiraju teškim i nepovratnim oštećenjima, pri čemu je regeneracija SŽS-a ograničena zbog ograničenog regenerativnog potencijala neurona. Ipak, regeneracija SŽS-a moguća je u embrijima, a regenerativni potencijal prestaje ubrzo nakon rođenja. Monodelphis domestica, sivi kratkorepi oposum, je tobolčar bez pravog tobolca čiji su mladunci rođeni vrlo nerazvijeni, ekvivalentno embrionalnom danu (E)13 kod štakora ili E12.5 kod miševa. Početni razvoj njihove kortikalne ploče događa se postnatalnog dana (P)3-5, dok gliogeneza počinje pri P18. Nadalje, oposumi imaju izvanrednu sposobnost potpune regeneracije svoje leđne moždine i do dva tjedna nakon rođenja, pri čemu njihova regenerativna sposobnost opada s porastom ne-neuronskih stanica. Stoga, oposumi su izvrstan životinjski model za proučavanje razvoja SŽS-a i regenerativnog potencijala in vitro. Primarne stanične kulture pripremljene iz korteksa ispitane su u različitim razvojnim dobima, P6 i P17. Stanični sastav primarnih kortikalnih staničnih kultura najbolje je karakteriziran kod P6 oposuma s imunofluorescentnom mikroskopijom. TUJ1+ neuronska populacija porasla je sa 60% pri in vitro danu (DIV)1 do vrhunca - 84% neurona pri DIV5, s padom na 44% pri DIV13. Astrociti sazrijevaju sa razvojem, od DIV5 do DIV13 njihova je populacija porasla sa 28% na 32% GFAP+ stanica. Regenerativni potencijal neuronskih i ne-neuronskih stanica unutar središnjeg živčanog sustava ispitan je s induciranom ozljedom u kortikalnim kulturama P6 i P17, simuliranom sa testom reza. P6 kulture su pokazale bolji regenerativni potencijal, pri čemu su neuroni regenerirali i izrasli aksone kroz mjesto reza, stvarajući konuse rasta različitih veličina ovisno o dobi. Astrociti su polarizirali, pri čemu su se njihovi izdanci izdužili okomito na mjesto reza ili su migrirali na mjesto, predstavljajući stvaranje glijalnog ožiljka. Primarne kortikalne kulture dobivene iz oposuma je nov izvor stanica sisavaca koji doprinosi in vitro istraživanjima.
Databáze: OpenAIRE
Externí odkaz: