Femur bone marrow from brain death deceased donors as source of human mesenchymal stromal cells for cell therapy.

Background: The use of a deceased donor (DD) as an alternative source of human mesenchymal stromal cells (hMSC) is promising, but has been little explored. This study evaluated the potential of femur bone marrow (FBM) from brain-death donors as a source of hMSC and compared this with hMSC from matched iliac crest bone marrow (ICBM). Methods: Sixteen donor-matched FBM and ICBM samples were processed from brain-death donors. We analyzed the starting material and compared cell yield, phenotypic profile and differentiation capacity of hMSC. Results: Neither the amount of nucleated cells per gram (14.6×106±10.3×106 from FBM vs. 38.8×106±34.6×106 from ICBM, P≥0.09) nor the frequency of CFU-F (0.0042%±0.0036% in FBM vs. 0.0057%±0.0042% in ICBM, P≥0.73) differ significantly from FBM or ICBM. Cell cultures from both sources were obtained and hMSC yields showed that there were no significant differences in hMSC obtained per gram of bone marrow (BM) when comparing femur with iliac crest samples. At passage 2, 12.5×106±12.9×106 and 5.0×106±4.4×106 hMSC per gram of BM were obtained from FBM and ICBM, respectively. FBM and ICBM hMSC express CD73, CD90, CD105, but not hematopoietic lineage markers [CD45, CD34, CD11, CD19 and isotype of HLA clase II (HLA-DR)]. HLA-A expression from both sources was clearly detected, while HLA-B was weakly expressed or undetectable and HLA-DR was undetectable. Cells from both sources were differentiated in vitro into osteoblasts, adipocytes and chondroblasts. Conclusions: To our knowledge, there are no previous studies evaluating BM from femur dead donors as a source of hMSC. Our findings confirm that it is feasible to expand cells from FBM from brain-death donors meeting in vitro characteristics of hMSC, making them a promising source for clinical translation.

Processing methods for human amniotic membrane as scaffold for tissue engineering with mesenchymal stromal human cells.

Tissue engineering is an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function. The aims of this work were to compare chemically and physically processed human Amniotic Membranes (hAM) and analyze the cytocompatibility and proliferation rate (PR) of two primary human mesenchymal stromal cell lines, from different sources and donor conditions seeded over these scaffolds. The evaluated hAM processes were: cold shock to obtain a frozen amniotic membrane (FEAM) with remaining dead epithelial cells, denudation of hAM with trypsin for 20/10 min (DEAM20/10) or treatment with sodium dodecyl sulfate to decellularized hAM (DAM). All samples were sterilized with gamma radiation. The selection of the treated hAM to then generate composites was performed by scanning and transmission electron microscopy and characterization by X-ray diffraction, selecting DEAM10 and FEAM as scaffolds for cell seeding. Two sources of primary human stromal cells were used, both developed by our researchers, human Dental Pulp Stem Cells (hDPSC) from living donors and human Mesenchymal Stromal Cells (hMSC) from bone marrow isolated from brain dead donors. This last line of cells conveys a novel source of human cells that, to our knowledge, have not been tested as part of this type of construct. We developed four in vitro constructs without cytotoxicity signs and with different PR depending on the scaffolds and cells. hDPSC and hMSC grew over both FEAM and DEAM10, but DEAM10 allowed higher PR.

Brain Death Diagnosis in Primary Posterior Fossa Lesions.

Background: New controversies have raised on brain death (BD) diagnosis when lesions are localized in the posterior fossa. Objective: The aim of this study was to discuss the particularities of BD diagnosis in patients with posterior fossa lesions. Materials and Methods: The author made a systematic review of literature on this topic. Results and Conclusions: A supratentorial brain lesion usually produces a rostrocaudal transtentorial brain herniation, resulting in forebrain and brainstem loss of function. In secondary brain lesions (i.e., cerebral hypoxia), the brainstem is also affected like the forebrain. Nevertheless, some cases complaining posterior fossa lesions (i.e., basilar artery thrombotic infarcts, or hemorrhages of the brainstem and/or cerebellum) may retain intracranial blood flow and EEG activity. In this article, I discuss that if a posterior fossa lesion does not produce an enormous increment of intracranial pressure, a complete intracranial circulatory arrest does not occur, explaining the preservation of EEG activity, evoked potentials, and autonomic function. I also addressed Jahi McMath, who was declared braindead, but ancillary tests, performed 9 months after initial brain insult, showed conservation of intracranial structures, EEG activity, and autonomic reactivity to "Mother Talks" stimulus, rejecting the diagnosis of BD. Jahi McMath's MRI study demonstrated a huge lesion in the pons. Some authors have argued that in patients with primary brainstem lesions it might be possible to find in some cases partial recovery of consciousness, even fulfilling clinical BD criteria. This was the case in Jahi McMath.