Optimized Clump Culture Methods for Adult Human Multipotent Neural Cells

Abstract

Adult human multipotent neural cell (ahMNC) is a candidate for regeneration therapy for neurodegenerative diseases. Here, we developed a primary clump culture method for ahMNCs to increase the efficiency of isolation and in vitro expansion. The same amount of human temporal lobe (1 g) was partially digested and then filtered through strainers with various pore sizes, resulting in four types of clumps: Clump I > 100 µm, 70 µm < Clump II < 100 µm, 40 µm < Clump III < 70 µm, and Clump IV < 40 µm. At 3 and 6 days after culture, Clump II showed significantly higher number of colonies than the other Clumps. Moreover, ahMNCs derived from Clump II (ahMNCs-Clump II) showed stable proliferation, and shortened the time to first passage from 19 to 15 days, and the time to 1 × 109 cells from 42 to 34 days compared with the previous single-cell method. ahMNCs-Clump II had neural differentiation and pro-angiogenic potentials, which are the characteristics of ahMNCs. In conclusion, the novel clump culture method for ahMNCs has significantly higher efficiency than previous techniques. Considering the small amount of available human brain tissue, the clump culture method would promote further clinical applications of ahMNCs.

Optimal Preclinical Conditions for Using Adult Human Multipotent Neural Cells in the Treatment of Spinal Cord Injury

“…First, ahMNCs isolated from patients with hemorrhagic stroke were successfully applied in a preclinical model of SCI. Previously, ahMNCs were cultured from temporal lobe epilepsy tissues, specifically, focal cortical dysplasia (FCD) type IIIa [ 9 , 10 , 11 ]. In FCD type IIIa, mild localized structural distortion of the cerebral cortex within the temporal lobe is accompanied by hippocampal sclerosis [ 19 , 20 ].…”

“…Brain tissue samples obtained from patients with hemorrhagic stroke were used in this study, and the primary culture of these specimens was completed as previously described [ 9 ]. Briefly, within 2 h of surgery, surgical specimens were weighed and minced and then partially digested using an enzyme solution containing 10 units/mL papain (Sigma, St. Louis, MO, USA), 0.1 mg/mL DNase I (Roche, Basel, Switzerland), and 4 mg/mL d, l-cysteine (Sigma) at 37 °C for 20 min.…”

“…Implanted NSCs replace lost neurons [ 2 , 3 ], create a favorable microenvironment supporting the regrowth of host axons [ 4 , 5 ], produce growth factors that protect damaged neural cells [ 6 , 7 ], and reform glial cells to support surviving neuron function [ 8 ]. We previously established an experimental protocol for the isolation and expansion of adult human multipotent neural cells (ahMNCs) from surgical brain samples [ 9 , 10 ]. These ahMNCs retain their self-renewal and differentiation capabilities allowing them to produce new neural cells, such as neurons and astrocytes, which are essential features of NSCs.…”

Prenatal stem cell therapy for inherited diseases: Past, present, and future treatment strategies

“…86 In contrast, NSC are more abundant in the developing early fetal brain and have been used in several clinical trials for treating neurodegenerative diseases. 87 To obtain a sufficient number of cells, multiple donors are required and hence with limited access to suitable donors and due to ethical considerations, scientists are considering using ESC or iPSC as an alternative source for replacement therapies for neurodegenerative diseases. 88 Recently a FDA-approved stem cell line (NSI-566) derived from first trimester fetal spinal cord was established and the use has led to promising results in phase I and II clinical studies of neurodegenerative diseases and spinal cord injuries.…”

Adult Human Multipotent Neural Cells Could Be Distinguished from Other Cell Types by Proangiogenic Paracrine Effects via MCP-1 and GRO

“…Previously, we have successfully isolated and cultured adult human multipotent neural cells (ahMNCs) from adult human temporal lobes [ 15 , 21 , 22 ]. They can be differentiated into neurons and astrocytes.…”