Adipose- and Bone Marrow-Derived hMSCs: What's the Difference?

INTRODUCTION:

Human Mesenchymal Stem/Stromal Cells, or hMSC, are key components of future therapeutics, engineered tissues, and medical devices and are currently in use in over 400 clinical trials (1). Bone marrow-derived MSC (hBM-MSC) have historically been the most widely used hMSC, but hMSC can be isolated from many tissues of the body including fat, umbilical cord blood, dental pulp, Wharton’s jelly, and peripheral blood. In recent years, human Adipose (or fat) tissue-derived MSC (hAD-MSC) are increasingly used in studies due to adipose tissue having a higher frequency of MSC than bone marrow and the relative ease of collection (2). You can find more information on hMSC by following these links:

•      What are MSCs
•      MSCs: The Workhorse of Regenerative Medicine
•      Widespread MSC Misconceptions
•      Using MSCs to Beat Cancer

A common MSC misconception is that MSC isolated from different tissues are equivalent. hAD-MSC and hBM-MSC, and cells from other tissues, can meet the “traditional” ISCT criteria to identify a cell as an MSC (3,4): adherence to plastic, characteristic surface marker expression profiles (positive for CD73, CD90, CD105; negative for CD34, CD45), and trilineage differentiation to fat, bone, and cartilage.  However, there is widespread acceptance that hMSC achieve their biologic and therapeutic effects in vivo by secreting many bioactive molecules (referred to as the hMSC secretome) that moderate a variety of processes including angiogenesis, immunosuppression, and overall “tissue repair” (5). Despite being similar overall, hMSC isolated from adipose and bone marrow display some differences in functional capabilities (2,6). For example, hBM-MSC are more robust in bone and cartilage differentiation than hAD-MSC and hAD-MSC are more efficient at stimulating angiogenesis than hBM-MSC (2,6,7).

We have recently been applying our manufacturing protocols to adipose-derived hMSC (our newest product) and would like to share some of the similarities and differences in function between hBM-MSC and hAD-MSC that we have observed when these cells are cultured in our media systems with our protocols.  Both populations of hMSC have been manufactured using our GMP-compatible and scalable manufacturing processes, with standardized procedures and with rigorous quality control.  By reporting the differential functional characteristics of these hMSC populations, we assist our customers in making more informed choices on the cell type best-suited to their application(s).

METHODS AND EXPERIMENTAL DESIGN:

Materials & Reagents:  Cell culture reagents, excluding RoosterBio materials, were purchased from Life Technologies, chemicals and reagents for kynurenine measurement were from Sigma, and cultureware was from Corning.  Two vials (1 million cells each) of hAD-MSC, representing two donors, were purchased from ZenBio, and used only for comparison. Other cell products used were RoosterBio hMSC products: Bone Marrow-derived MSC (hBM-MSC, part # MSC-001, MSC-003) and Adipose-derived MSC (hAD-MSC, part # MSC-020, MSC-021). Cells were cultured in RoosterBio High Performance Media (part # KT-001) or DMEM + 10% FBS

Methods: All methods for the analyses shown below are documented under RoosterBio’s Quality Control systems.  For more information, please contact us at info@roosterbio.com.  Detailed methods for priming hMSC can be found in a previous blog post here.

RESULTS:

NIST Workshop Aims to Educate on Improving Confidence in Measurements Critical for Cell Therapy Products

We are always looking for ways to enable the commercialization of Cell-based Therapies and Technologies.  The ultimate success of the field is dependent on the convergence of several technology fields, and one that isn't given sufficient attention is the Measurement Sciences (or metrology).  The National Institute of Standards and Technology (NIST) has a Biosystems and Biomaterials Division that has several projects focused on their mission of "Building Confidence in Biological Measurements", and they have several people that are working to advance measurements in regenerative medicine.

The NIST BioSystems and Biomaterials Division has several projects related to stem cells and regenerative medicine.

On May 11&12, NIST will be holding a workshop focused on Measurement Assurance for Cell Therapy Products.  The Registration for the workshop is open and is limited to the first 100 registrants, and the Agenda (below) has speakers from Industry, the FDA, NIST, as well as several breakout workshops to focus on some of the analytics that are the low hanging fruit to bring standardization too.

The adage of "Measure Twice, Cut Once" only holds true if you have robust and precise methods and assays, and solid reference standards for which everyone can compare.  Our previous posts on Regenerative Medicine Standardization, and of course the great content on this at the Stem Cell Assays blog are good places for background reading for those interested.

An Open Letter to the Builders of the Cell-based BioEconomy

Dear Stem Cell Pioneer:

February marks the one year anniversary of RoosterBio shipping our first stem cell products to our valued customers, and I personally am very excited for the coming year ahead. We look forward to delivering even more high quality stem cells to people like you that are doing amazing things.

Looking forward into 2015 and beyond, I want to make sure we are staying true to our mission: to greatly increase the availability and accessibility of stem cell technology to researchers and product developers across the globe – and that we are committed to our vision of accelerating the pace of product development in the cellular therapy, bioprinting and tissue engineering markets.  I am hoping to focus our efforts to making sure that we are moving your discoveries and developments forward faster than anticipated.  This will not just be a win for our customers and RoosterBio, but for the entire Regenerative Medicine field.

In 2014 after launching our hMSCs in the unprecedented product format of 10 million cell vials to glowing reviews, we quickly implemented our Starter Kits and Working Cell Bank formats based on your feedback.  These new formats allow for accelerated testing, performance verification, and standardized small scale experimentation with reproducible outcomes.  We also initiated multiple collaborations with leaders in the tissue engineering and biofabrication fields – which has led to multiple conference posters, presentations, and soon to be submitted publications.

We also find ourselves at the precipice of a boom in biofabrication technologies, and we consider this the beginning of the Golden Age of Tissue Engineering.  I anticipate great progress will be made at an increasingly rapid pace.  Now that many of the tools required for bioprinting are becoming “democratized” (simpler, less expensive, more accessible) such as 3D BioPrinters, biomaterials, and primary cells – laboratories can get up and running in a matter of weeks with limited initial resources, something that would have taken months to years and extensive capital in the past.  We are at a special time, and the entire field will be accelerating forward at a rapid pace, making biofabrication truly an exponential medical technology.

2015 will truly be an exciting year for RoosterBio.  We will be participating in a Stem Cell Manufacturing Training Program, helping to organize several conferences on Cell Therapy BioProcessing and BioPrinting, exhibiting at multiple conferences, as well are contributing to initiatives such as the Georgia Tech Cell Manufacturing Consortium and the NIST Workshop on Strategies to Achieve Measurement Assurance for Cell Therapy Products.  The primary motivation behind these initiatives are to make sure that we are driving forward our vision and delivering on our mission.

None of this would be possible without the hard work and dedication of the entire RoosterBio team, as well as the support that we are getting from you, our valued customers.  Please continue to join us on our journey as we accelerate the development of the Cell-based BioEconomy.

All the best from Frederick, Maryland.


Jon A Rowley
Chief Executive & Technology Officer
RoosterBio Inc.

The Rise of BioFabrication and BioPrinting in Tissue Engineering & Regenerative Medicine – notes from TERMIS 2014 Annual Meeting

RoosterBio participated in the annual Tissue Engineering and Regenerative Medicine International Society’s Annual Meeting of the Americas chapter (or TERMIS-AM for short) in Dec 2014.  You can find a lot of content on the meeting at the conference website where you can download the program for free, as well as read the published abstracts in the journal Tissue Engineering.  The final registration numbers for the 2014 TERMIS-AM conference in DC was 842 (about a 7% increase over last year’s conference).  There were 30 countries represented at the conference, with a total of 202 oral presentations and 338 poster presentations (Stats from Sarah Wilburn at the TERMIS head office).  We are looking forward to the 2015 TERMIS World Congress, which will be in Boston in early September, 2015.

There were two striking trends that were gleaned from the conference that I wanted to outline over a couple of blog posts.  First, there was a noticeable rise in the number (and quality) of the Biofabrication-related talks and posters (this blog post will focus on this).  The second trend to note was the rise in Product Development content at the 2014 meeting – and this will be the focus of a subsequent blog post.  Interestingly, the intersection of these two topics (manufacturing process technologies and product development) has traditionally been crucial for the successful commercialization of high tech products, including biopharmaceuticals (see recent HBR article by Pisano and Shih here).  

Our favorite booth (after the RoosterBio booth, or course) was BioBots', who were
showing off the beta version of the BioBot Rapid 3D Bio-Prototyper.

The Rise of BioFabrication and BioPrinting in Tissue Engineering

TERMIS has always been a great conference for academic Tissue Engineering technologies.  The major comment that I always heard from fellow industrialists was just how “academically” focused the conference was.  Meaning that the

Welcome to the Golden Age of BioPrinting, Tissue Engineering and BioFabrication

A"Golden Age" is defined as a period of time in a field where "great tasks are accomplished."  The ancient Greek philosopher Hesiod initially coined this phrase, and I think if he were alive today, he would agree with us that we are in a special time of technology convergence where innovations and advancements are progressing at an accelerating rate.  The fields of Tissue Engineering and Regenerative Medicine are benefiting from these rapid technology advancements.



We are now at the beginnings of the Golden Age of BioFabrication.  The last 20 years has seen steady progress in the Tissue Engineering field, but the cost and time it has taken to develop products based on these technologies has been prohibitive.  Thus, only the best funded labs have been able to perform this very expensive R&D.  Within the last year, products such as high volume stem cells (via RoosterBio) and low cost bioprinters (from our collaborators BioBots) have been coming to market and dramatically reduce the cost, the time, and the complexity to fabricate three dimensional biological structures that are the precursors to tomorrow's tissue engineered products.  By removing the technology and cost barriers and democratizing biofabrication technology, more labs can now afford to do the applied R&D, allowing more work to be accomplished faster, completely changing the equation of how labs function.  This is accelerating the development of this entire field.

 Walter Isaacson makes the point over and over in his new book The Innovators that collaboration between people and groups with complementary skill sets is essential to innovation and technology progress.  We, at RoosterBio, have always said that communication platforms (such as social networks, conferences, biohacker spacers, blogs, and journals) are also critical for those in a field to share knowledge and experiences - further progressing the thought convergence.  This February 9th and 10th in Boston is a focused conference on Tissue Engineering and BioPrinting that SelectBio is hosting.  The top researchers, thought leaders, and product developers in the field will be presenting cutting edge research, technology development, and commercialization strategies.  We hope to see you there.

Priming of hMSCs to Improve Potency

By Iain Farrance, Priya Baraniak, and Jon Rowley. RoosterBio.

In this blog, we will present internal data and information on priming RoosterBio’s bone marrow derived human MSCs (hMSC) with pro-inflammatory molecules and the impact of these priming protocols on hMSC immunomodulatory function and angiogenic cytokine secretion.

INTRODUCTION:

Human Mesenchymal Stem/Stromal Cells, or hMSCs, are key components of future therapeutics, engineered tissues, and medical devices. There are currently over 400 clinical trials investigating hMSCs as therapies (1). The trials have produced some promising results, with hMSCs generally deemed safe, and in some cases effective (2). It is believed that these versatile cells achieve their biologic and therapeutic effects by secreting a plethora of biomolecules (referred to as the MSC secretome) that moderate a variety of processes including angiogenesis, immunosuppression, and overall “tissue repair” (3-6). As the secretome is one of the likely Mechanisms of Actions (MOA) of hMSC therapies, there is a significant amount of recent work on engineering hMSC preparations to enhance secreted factors by genetic modification, by culture strategies, or by engineering the hMSC microenvironment (5, 7-11). In addition, a recent ISCT paper (12) advances the concept of “priming” hMSCs by exposing the cells to pro-inflammatory cytokines prior to implantation.

Thus, priming of hMSCs can have two primary purposes:

1. To assess human MSC preparations in vitro as recommended by the ISCT and the FDA (12, 13), and
2. To enhance hMSC potency (survival, immunosuppression, homing) prior to implantation (8, 9, 14).

As part of our standard quality control (QC) testing, RoosterBio analyzes the immunosuppressive capability of our cell lots through priming with IFN-γ. The hMSC response (i.e. immunomodulatory potential) is reported as a measure of indoleamine 2,3-dioxygenase (IDO) activity, determined by measuring the amino acid kynurenine in the culture supernatant.  The IDO enzyme converts L-tryptophan to N-formylkynurenine (or kynurenine), an immunosuppressive molecule that acts as an inhibitor of immune cell proliferation - including T cells (12, 15, 16).  Testing every hMSC lot for inducible IDO activity provides a quality assurance that the cells we release have some level of functional potency as it relates to immunomodulation– which we consider a key quality attribute of hMSCs.

While researchers are beginning to implement testing of hMSC preparations for inducible IDO activity prior to implantation, few are looking at the impact of priming on other hMSC functions.  Here, we present information on priming of RoosterBio’s hMSCs with IFN-γ ± TNF-α across multiple lots and donors and the impact of such treatment on hMSC IDO activity and angiogenic cytokine secretion.  The goal of this blog post is to demonstrate that priming has impacts on several functional properties of hMSCs, and that researchers should consider priming regimens to (a) understand the potency of their specific cell products, especially in inflammatory environments, and (b) to potentially increase potency of these cell products upon therapeutic administration.

METHODS & EXPERIMENTAL DESIGN:

Materials & Reagents

Cell culture reagents were purchased from Life Technologies, chemicals and reagents for kynurenine measurement were from Sigma, and cultureware was from Corning.  Other products are: Bone Marrow-derived human MSCs (BM-hMSC, part # MSC-001, RoosterBio) and RoosterBio High Performance Media kit (part # KT-001).

Table 1: Experimental design.

hMSC Priming

Current Bottlenecks in MSC Research: MSC Misconceptions - Part II

 http://andreyev.com.au/wp-content/uploads/Misconceptions.jpg

We blogged recently about Mesenchymal Stem/Stromal Cell (MSC) Misconceptions that are holding the translational cell therapy field back, as identified by Donald Phinney and Luc Sensebé.  Since we have come to market with our own hMSC product lines, we have spoken with hundreds of MSC researchers and engineers, and we have compiled our own set of misconceptions that we think build off of Dr. Phinney’s and Dr. Sensebe’s initial concept.  This blog post is to share some of the market-based feedback that we have received.

To the list that was published in Cytotherapy, we would like to contribute the following list to the conversation:

1. Tracking MSC passage number is an accurate and reliable means of tracking cell age and standardizing experimental workflow

In many research laboratory environments, cellular age is most often tracked by the number of times a cell has been passaged; however, Passage Number is quite imprecise and not very acceptable as one gets into regulated environments such as translational clinical activities.  It is generally accepted that tracking the Population Doubling Level (PDL) or Cumulative Population Doublings (CPD) of primary cells is a best practice on understanding cellular age in vitro.   Since it is well documented that PDL impacts hMSC function (see here, here and here), in order to drive consistency into experiments, it has become a best practice to perform experiments or develop products with cells in a consistent range of population doublings where the cell function of interest is still robust.  Furthermore, regulatory agencies are beginning to require reporting of PDLs, or at least cell seeding and harvest densities, for primary cells intended for therapeutic use.  In an effort to drive adoption of PDL tracking and reporting, we’ve created a Best Practices Educational Powerpoint, and free, easy-to-use PDL calculator worksheet we’re happy to share with colleagues.  For your copy, just email us at info@roosterbio.com or subscribe to our blog!

2. Performing experiments with one MSC donor and/or lot is adequate for publication and moving forward with pre-clinical studies

Despite indications of clinical effectiveness of MSCs, there is repeated news of the failure of high-profile MSC trials to demonstrate efficacy in a number of therapeutic applications.  It has been suggested that the large amount of intra- and inter-donor variability in the MSC populations used in these trials may be responsible for their falling short of expectations despite highly encouraging in vitro and in vivo pre-clinical data.  Thus, to ensure the robust production of functional MSC products over a range of applications, experiments should be conducted and systems validated with MSCs from several donors.  It has been reported that best practices to qualify a manufacturing process should include “at least 3-5 donors”, and it is likely that proper Validation will require many more, and that donor selection may be required (i.e. not every donor will work in the manufacturing process). This is why we, at RoosterBio, believe in providing a number of donor MSC lots, ranging in age and sex, for use in our customer’s research and development experiments.

3. MSCs accelerate cancer…..MSCs can combat cancer

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.

How Quickly are Cell-based Products Really Developing? Thoughts from the IBC Cell Therapy Bioprocessing Conference

Last week was the 4^th Annual IBC Cell Therapy Bioprocessing Conference.  IBC (home of the BioProcess International conference) was the first conference organizer to dedicate a focused meeting on Cell Therapy Manufacturing Technologies 4 years ago.  Since the first conference in 2011, the growth in the field, and the conference, has been amazing.  The attendance has grown from less than 90 in year 1 to over 200 this year.  The content has also evolved heavily over the last 4 years, demonstrating a high level of sophistication and maturity in a field that seems “early stage” to those looking in from the outside.  The talks this year increased in the amount and quality of data presented. Topics included the impact of automation on the simplification, streamlining, and cost reduction of autologous therapies, the use of Quality by Design (QbD) in bioreactor scale-up and analytical development, advances in tissue engineering and biofabrication techniques, and even 2 year data on marketed products.   Phil Vanek, the General Manager of GE Healthcare’s Cell Therapy business, summed it up during his talk where he stated that: GE is interested in 1) big problems, 2) compelling clinical data, and 3) opportunities for “industrialization”, and “Cell Therapy/Regenerative Medicine has all three”.

Various cell manufacturing and processing devices seen throughout the exhibits at IBC's
4th Annual Cell Therapy BioProcessing Conference - No BioPrinters (yet!)

There are many signs that the Cell Therapy field is moving much faster than the protein therapeutics field before it and demonstrating rapid progress.  What we have here is a traditional case of  “standing on the shoulders of giants”, which has been paraphrased on Wikipedia as "discovering truth by building on previous discoveries”.