title: Sequential cultivation of human epidermal keratinocytes and dermal mesenchymal like stromal cells in vitro.
abstract: Human skin has continuous self-renewal potential throughout adult life and serves as first line of defence. Its cellular components such as human epidermal keratinocytes (HEKs) and dermal mesenchymal stromal cells (DMSCs) are valuable resources for wound healing applications and cell based therapies. Here we show a simple, scalable and cost-effective method for sequential isolation and propagation of HEKs and DMSCs under defined culture conditions. Human skin biopsy samples obtained surgically were cut into fine pieces and cultured employing explant technique. Plated skin samples attached and showed outgrowth of HEKs. Gross microscopic examination displayed polygonal cells with a granular cytoplasm and H&E staining revealed archetypal HEK morphology. RT-PCR and immunocytochemistry authenticated the presence of key HEK markers including trans-membrane protein epithelial cadherin (E-cadherin), keratins and cytokeratin. After collection of HEKs by trypsin-EDTA treatment, mother explants were left intact and cultured further. Interestingly, we observed the appearance of another cell type with fibroblastic or stromal morphology which were able to grow up to 15 passages in vitro. Growth pattern, expression of cytoskeletal protein vimentin, surface proteins such as CD44, CD73, CD90, CD166 and mesodermal differentiation potential into osteocytes, adipocytes and chondrocytes confirmed their bonafide mesenchymal stem cell like status. These findings albeit preliminary may open up significant opportunities for novel applications in wound healing.
authors.affiliation: School of Regenerative Medicine, Manipal University Branch Campus, Bangalore, 560065, India.
authors.forename: Shyam
authors.initials: S
authors.lastname: Mahabal
authors.affiliation: Department of Marine Biotechnology, AMET University, Kanathur, Chennai, 603112, India.|Genes N Life Health Care Pvt. Ltd, Punjagutta, Hyderabad, 500082, India.
authors.forename: Vijay Bhaskar Reddy
authors.initials: VB
authors.lastname: Konala
authors.affiliation: School of Regenerative Medicine, Manipal University Branch Campus, Bangalore, 560065, India.
authors.forename: Murali Krishna
authors.initials: MK
authors.lastname: Mamidi
authors.affiliation: School of Regenerative Medicine, Manipal University Branch Campus, Bangalore, 560065, India.
authors.forename: Mohammad Mahboob
authors.initials: MM
authors.lastname: Kanafi
authors.affiliation: Department of Pathology, Manipal Hospital, 98, Rustom Bagh, Old Airport Road, Bangalore, 560017, India.
authors.forename: Suniti
authors.initials: S
authors.lastname: Mishra
authors.affiliation: Department of Dermatology, Manipal Hospital, 98, Rustom Bagh, Old Airport Road, Bangalore, 560017, India.
authors.forename: Krupa
authors.initials: K
authors.lastname: Shankar
authors.affiliation: School of Regenerative Medicine, Manipal University Branch Campus, Bangalore, 560065, India.
authors.forename: Rajarshi
authors.initials: R
authors.lastname: Pal
authors.affiliation: School of Regenerative Medicine, Manipal University Branch Campus, Bangalore, 560065, India. rr.bhonde@manipal.edu.
authors.forename: Ramesh
authors.initials: R
authors.lastname: Bhonde
keywords: Human skin; Mesenchymal stem cells and co-culture; Primary keratinocytes