Current Bottlenecks in MSC Research: Widespread MSC Misconceptions

We blogged several months ago about bottlenecks in the bioprocessing of Mesenchymal Stem Cells that are impeding their clinical translation.  While the development of robust and scalable manufacturing methods, reduced cost of goods, and implementation of solid Quality Systems are all necessary for increased clinical use of MSCs, there are also current misconceptions surrounding MSCs, rooted in decades-old science, that are holding the field back.  I recently came across a paper from last year on this topic and decided to put it forth for discussion here – with a few targeted opinions from us scientists at RoosterBio.  It is my hope that you’ll provide your own opinions on the misconceptions detailed here, as well as your suggestions on other misconceptions you think could be holding the MSC field back.

The authors of the 2013 Cytotherapy Paper: Mesenchymal stromal cells: misconceptions and evolving concepts, Donald Phinney and Luc Sensebé, identify six major misconceptions that have persisted over the years, despite widely-accepted paradigm shifts on MSC nature and function.  Here, I will summarize four of these misconceptions and add our take to them.

Four of the misconceptions identified by Phinney and Sensebé:

1. MSCs isolated from different tissues are equivalent

Initially isolated from bone marrow in the 1950s, MSCs were then discovered in adipose tissue, and have since been found in a number of tissues including, but not limited to: Wharton’s jelly, umbilical cord blood, placenta, amnion, and dental pulp.  While MSCs from all these sources are somewhat similar in surface profile marker expression, phenotype, and gene expression profiles, their functionality, in terms of differentiation potential, immunomodulatory activity, and paracrine factor secretion, can vary widely depending on the tissue of origin.  ** Given that MSCs from a single tissue and donor are not equivalent (see below), it comes as no surprise that MSCs from different tissues vary in function! **

Rapid and Economic Generation of hMSC Spheroids for Macroscopic Tissue Biofabrication

Mesenchymal stem cells (MSCs) aggregated into three-dimensional (3D) cellular spheroids are a potent configuration for cell therapy and tissue engineering research and product development (5), and cellular spheroids are a preferred format for many bioprinting applications (10).  Cellular spheroids are essentially micro-tissues that can be manufactured as standardized “living materials” with certain controllable, measurable, and evolving material properties (10). Studies have shown that MSC aggregation into spheroids yield improved in vitro biological functionality over MSCs grown as 2D monolayer; likely due to the 3D tissue-like structure resembling the native configuration of cells in vivo with a microenvironment that allows for direct cell-cell signaling and cell-matrix interactions. MSC spheroids demonstrate enhanced cartilage, bone, and fat differentiation, as well as increased paracrine factor secretion over 2D MSC cultures (1-7). In vivo administration of hMSC spheroids has also showed enhanced therapeutic properties in pre-clinical models of myocardial infarction, bone and cartilage repair, and limb ischemia (3, 5, 8).  

Traditionally, aggregates were formed using suspension culture in spinners or shake flasks or in hanging drop cultures (5). The advancement of technologies has allowed one to quickly and easily generate large numbers of spheroids consistent in size and shape using forced aggregation in micro-wells (AggreWells, Stem Cell Technologies), or using liquid handling automation and 96 or 384 well hanging-droplet plates. There are also tools available that allow researchers to mold micro-tissues into interesting shapes such as rods, toroids, honeycombs, or whatever one can dream up (12-13).  While there are multiple methods for creating hMSC micro-tissues, the biggest challenge is reproducibly growing up sufficient hMSCs to create enough spheroids to start an experiment.  For example, if a researcher needs 10,000 spheroids with an average of 1000 cells per spheroid, then he/she will need at least 10 million cells to begin the experiment, which can take weeks to grow (see process flow diagrams below).  If he/she wishes to use 5000, or 10,000 cells per spheroid, then he/she will need 50 million or 100 million cells for his/her experiment.  This volume of cells has traditionally been very costly and time consuming to generate.

This Application Blog Post will provide a simple protocol to rapidly and economically generate tens of millions of high quality hMSCs so that researchers can minimize their time spent on routine cell culture and maximize their effort on performing hMSC spheroid-based experiments.