Mesenchymal Stem Cells from Umbilical Cord Promote Repair After Heart Attacks in Minipigs

The umbilical cord contains a major umbilical vein and an umbilical artery, but these blood vessels are embedded in a gel-like matrix called “Wharton’s jelly.” Wharton’s jelly is home to a population of mesenchymal stem cells that have peculiar properties.

You might first say, “what on earth is a mesenchymal stem cell?” Fair enough. Mesenchymal stem cells were first discovered in bone marrow. In bone marrow, mesenchymal stem cells (MSCs) do not make blood cells; that;’s the job of the hematopoietic stem cells (HSCs). MSCs in bone marrow serve an important support role for HSCs in bone marrow. Traditionally, MSCs have the capacity to differentiate into fat cells, bone cells, and cartilage cells. However, further has shown that MSCs can also form a variety of other cell types as well if manipulated in the laboratory. MSCs also express are characteristic cadre of cell surface proteins (CD10, CD13, CD29, CD44, CD90, and CD105 for those who are interested).

MSCs, however, are found in more places that just bone marrow. As it turns out, MSCs have been found in fat, muscle, liver, tendons, synovial membrane (the membranes that surround joints, skin, and so on. Some scientists think that every organ in the body may harbor a MSC population. Furthermore, these MSC populations differ in the genes they express, their capability to differentiate into different cell types, and their cell surface proteins (see this article on this website for a rather exhaustive foray into this topic).

Now that you are more savvy about MSCs, Wharton’s jelly contains a MSC population, but this population seems to have a younger profile than MSCs from other parts of the body.  They are more plastic and more invisible to the immune system than other types of MSCs.  For that reason,  they might be good candidates for treating a sick heart after a heart attack.  A recent paper by Wei Zhang and others from the TEDA International Cardiovascular Hospital and the Tianjin Medical Cardiovascular Clinical College examined the ability of MSCs from the Wharton’s jelly of human umbilical cords to heal the hearts of minipigs after a heart attack.  Oh, before I forget – this paper was published in the journal Coronary Artery Disease.

Twenty-three minipigs were subjected to open-heart surgery and given heart attacks.  Then the pigs were divided into three groups, a control group, a group that received injections of saline into their hearts, and a third group that received injections of 40 million human Wharton’s jelly derived MSCs into the region of the infarct.  The animals were sewn up and given antibiotics to prevent infection.

Six weeks after surgery, each animal was examined by means of Technetium-sestamibi myocardial perfusion imaging, and electrocardiography. For those who do not know what Technetium-sestamibi myocardial perfusion imaging is for, it works like this.  Cardiolite is the trade name of a large, fat-soluble molecule that flows through the heart in a fashion proportion to the blood flow through the heart muscle.  Single photon emission computed tomography or SPECT is used to detect the Cardiolite.    Areas of the heart without blood flow are the regions damaged during the heart attack.  Therefore, this technique is extremely useful to determine the area of damage in the heart.


After the animals were examined, they were put down and their hearts were extracted, sectioned, and stained for areas or cell death, and the areas where the injected stem cells resided.  All injected stem cells were labeled before injection so that they were easily detectable.

The results were clear.  The heart injected with MSCs from umbilical cord did not show any decrease in ejection fraction, whereas the other two groups showed an average reduction in injection fraction of around 10%.  In fact the stem cell-injected hearts showed an average 1 % increase in ejection fraction.  The blood flow in the hearts was even more different.  blood flow is measured as a ratio of dead heart tissue to total heart tissue.  The control of saline-injected hearts had an average ratio of about 4%, whereas the stem cell-injected hearts had a slightly negative percentage.  This is a significant difference.   Echocardiography confirmed that the wall thickness of the stem cell-injected hearts was significantly thicker than the walls of the control or the saline-injected hearts; some 14 times thicker!!

When the dissected hearts were examined, the MSC-injected hearts had lots of stem cells still in them.  The cells not only survived, but, according to Zhang and his colleagues, differentiated into heart muscle cells.  Their rationale for this conclusion is three-fold – the cells had the same shape and form or native heart muscle cells, they expressed heart specific Troponin T and vWF proteins, and electrically coupled with other heart muscle cells by expressing connexin.  Connexin is a protein that traverses the membranes of two closely apposed cells and forms small pores between two cells that allows the exchange of SMALL molecules such as ions, ATP, and things like that.  These connexin constructed pores are called “gap junctions” and they are the reason heart muscle cells work as a single unit, since any electrochemical change in one cell immediately spreads to all other nearby, connected cells.

gap junctions

As much as I would like to believe Zhang and his colleagues, I remain skeptical that these cells differentiated into heart muscle cells.  I say this because MSCs can be differentiated in culture to form cells that look and act like heart muscle cells.  These cells will even express some heart-specific genes.  However, they lack the calcium handling machinery of true heart muscle cells and do not function as true heart muscle.  To convince that these Wharton jelly MSCs truly are heart muscle cells, they will need to show that they contain heart specific calcium handling proteins (see Shake JG, Gruber PJ, Baumgartner WA et al. Ann Thorac Surg 2002;73:1919–1925; Davani S, Marandin A, Mersin N et al.  Circulation 2003;108(suppl 1):II253–258; Hou M, Yang KM, Zhang H et al. Int J Cardiol 2007;115:220 –228).  If they can show this, then I will believe them.

However, there are two findings of this paper that are not in doubt.  The number of blood vessels in the hearts of the MSC-treated animals far exceeded the number found in the control or the saline-treated hearts (3-4 times the number of blood vessels).  Therefore, the Wharton’s jelly MSCs induced lots and lots of blood vessels.  Many of these blood vessels contained labeled cells, which shows that the MSCs differentiated into endothelial and smooth muscle cells, Also, the Wharton’s jelly MSCs clearly induced resident cardiac stem cell (CSC) populations in the hearts of the minipigs, since several cells that expressed CSC surface molecules were found in the heart muscle tissue.  Previous work by Hatzistergos and others showed that MSCs induce the endogenous CSC population and this is one of the ways that MSCs help heal ailing hearts (Circulation Research 2010 107:913-22).

Zhang’s paper is interesting and it shows that Wharton’s jelly MSCs are safe and efficacious for treating the heart after a heart attack.  Also, none of the minipigs in this experiment were treated with drugs to suppress the immune system.  No immune response against the cells was reported.  Therefore, the invisibility of these cells to the immune system seems to last, at least in this experiment.

Directly Programming Skin Cells to Become Blood-Making Stem Cells

Within our bones lies a spongy, ribbon-like material called bone marrow.  Bone marrow is home to several different populations of stem cells, but the star of the stem cell show in the bone marrow are the hematopoietic stem cells or blood-making stem cells.   When a patient receives a bone marrow transplant these are the stem cells that are transferred, take up residence in the new bone marrow, and begin making new red and white blood cells for the patient.  Because bone marrow is such a precious commodity from a clinical standpoint, finding a way to make more of it is essential.

Hematopoiesis from Pluripotent Stem Cell

A new report from scientists at Mt Sinai Hospital in New York suggest that the transfer of specific genes into skin fibroblasts can reprogram mature, adult cells into hematopoietic stem cells that look and function exactly like the ones normally found within our bone marrow.

A research team at the Icahn School of Medicine at Mount Sinai led by Kateri Moore screen a panel of 18 different genes for their ability to induce blood-forming activity when transfected into fibroblasts. Kateri and others discovered that a combination four different genes (GATA2, GFI1B, cFOS, and ETV6) is sufficient to generate blood vessel precursors with the subsequent appearance of hematopoietic stem cells. These cells expressed several known hematopoietic stem cell surface proteins (CD34, Sca1 and Prominin1/CD133).

Reprogramming of fibroblasts to HSCs

“The cells that we grew in a Petri dish are identical in gene expression to those found in the mouse embryo and could eventually generate colonies of mature blood cells,” said Carlos Filipe Pereira, first author of this paper and a postdoctoral research fellow in Moore’s laboratory.

The combination of gene factors that we used was not composed of the most obvious or expected proteins,” said Ihor Lemischka, a colleague of Dr. Moore at Mt. Sinai Hospital.  “Many investigators have been trying to grow hematopoietic stem cells from embryonic stem cells, but this process has been problematic.  Instead, we used mature mouse fibroblasts, pick the right combination of proteins, and it worked.”

According to Pereira, there is a rather critical shortage of suitable donors for blood stem cells transplants.  Bone marrow donors are currently necessary to meet the needs of patients suffering from blood diseases such as leukemia, aplastic anemia, lymphomas, multiple myeloma and immune deficiency disorders.  “Programming of hematopoietic stem cells represents an exciting alternative,” said Pereira.

“Dr. Lemischka and I have been working together for over 20 years in the fields of hematopoiesis and stem cell biology,” said Kateri Moore.  “It is truly exciting to be able to grow these blood forming cells in a culture dish and learn so much from them.  We have already started applying this new approach to human cells and anticipate similar success.”

Clinical-Scale NK Cells for Cancer Therapy Made from Pluripotent Stem Cells

Dan Kaufman’s laboratory has done it again. The Kaufman laboratory at the University of Minnesota in collaboration with scientists from MD Anderson Cancer Center in Houston, Texas have designed a protocol to make natural killer cells from embryonic stem and induced pluripotent stem cells.

Natural killer cells provide a very important contribution to the innate immune response. These cells produce molecules called cytokines and they also kill virally infected cells and malignant cells. NK cells are unique among the cells of the immune system in that they have the ability to recognize foreign, infected or stressed cells in the absence of antibodies and Major Histocompatibility Complex proteins (the cell surface proteins that act as bar codes used by the immune system uses to determine if a cell is yours or not yours). Therefore, NKs typically work faster than the rest of the immune system.

Natural killer cells or NK cells have been used to treat patients with refractory cancers. Unfortunately, a major problem with using NK cells is growing a sufficient quantity of cells for therapy. Using pluripotent stem cells to make NK cells is an intriguing possibility, but the protocols for differentiating NK cells from embryonic stem cells (ESCs) is tedious and inefficient. However, the Kaufman laboratory has provided a much more efficient and straight-forward way to derive NK cells, thus allowing for the production of clinical scale quantities of NK cells.

The Kaufman lab protocol involves first deriving embryoid bodies from ESCs or induced pluripotent stem cells (iPSCs), which are made from adult cells through genetic engineering techniques that causes the cells to de-differentiate into ESC-like cells known as iPSCs. Embryoid bodies are three-dimensional aggregates of pluripotent stem cells that assume a kind of spherical shape and have a variety of cells differentiating into a wide range of cell types. Embryoid bodies can contain beating heart muscle, neural-type cells, blood progenitors cells, and even muscle or bone cells in their interiors in a haphazard arrangement. Forming embryoid bodies or EBs from cultured ESCs or iPSCs is rather easy, but controlling the differentiation of the cells in the EBs is quite another matter.

embryoid bodies
embryoid bodies

Kaufman and others discovered that if the EBs were incubated with artificial antigen-presenting cells that expressed a surface-bound version of the protein IL21 (interleukin 21) plus a cocktail of cytokines, these pluripotent stem cells could efficiently form NK cells.

Functional assays of the NK cells differentiated from ESCs and iPSCs easily showed that the NK cells for functional in every way and expressed all the cell surface molecules characteristic of NK cells. Furthermore, all ESC and iPSC lines examined were able to make NK cells, but the efficiency with which they made them different rather widely.

In conclusion, Kaufman and others state in their paper, “our ability to now produce large numbers of cytotoxic NK cells means that prospect hESC- and iPSC-derived hematopoietic products for diverse clinical therapies can be realized in the not-too-distant future.” For some cancer patients, that day cannot come soon enough.

Maintaining the Reservoir of Blood Cell-Making Bone Marrow Stem Cells

Stowers Institute for Medical Research fellow Linheng Li has discovered a mechanism by which blood cell-making stem cells maintain a reservoir of cells that can replenish these stem cells under stressful conditions. His paper greatly advances our knowledge of these stem cells.

Li and his colleagues have identified two cell surface molecules that keep mouse hematopoietic stem cells from proliferating when they are not needed. Not all adult stem cells are created equally. Some are busy repairing damaged tissues while others held in reserve to replenish those stem cells that have worn themselves out healing other tissues. Blood cell-making stem cells or hematopoietic stem cells are no different. some are held in reserve while others are actively making new blood cells. What tells some to grow and others to lay low?

Li’s group found that two cell surface proteins known as Flamingo and Frizzled8 regulate hematopoietic stem cell (HSC) proliferation. The activity of these two proteins helps maintain a reserve pool of HSCs in mouse bone marrow. In doing so, they help control the delicate balance between long-term maintenance of stem cell populations in the bone marrow, and the requirements of ongoing tissue maintenance and regeneration.

Li explains: “HSCs daily produce billions of blood cells via a strict hierarchy of lineage-specific progenitors. Identifying the molecular signals that allow HSC populations to sustain this level of output over a lifetime is fundamental to understanding the development of different cell types, the nature of tumor formation, and the aging process. My hope is that these insights will help scientists progress towards new therapies for diseases of the blood.”

Currently, work in Li’s laboratory and other labs has provided a working model that predicts that HSCs consist of a population that only divides a few times a year and sit, essentially, in reserve. These reserve cells only become activated when they need to replace other active HSCs that have been damaged by the daily war-and-tear or in response to injury or disease that greatly depletes the blood supply (Li J. Exp Hematol. 2011;39(5):511-20 & Ezoe S, et al., Cell Cycle. 2004;3(3):314-8). Missing from this working model is how the reserve and active populations of HSCs are maintained and regulated.

Both of these HSC populations exist in specialized locations within the bone and these locations or “niches” provide external cues to regulate the proliferation of the respective HSC population. Approximately 90% of all HSCs frequently divide and are found in the central marrow near blood vessels and endothelial and perivascular cells. Reserve HSCs tend to be located in the spongy part of the bone (trabecular bone) at the end of long bones. Reserve HSCs are usually very intimately associated with immature versions of bone-making cells (pre-osteoblasts). In fact, the reserve HSCs tend to be in contact with the pre-osteoblasts and this contact is extremely important from a regulatory perspective.

In Li’s lab, graduate student Ryohichi Sugimura examined Flamingo and Frizzled8 on the surface of reserve HSCs. Both of these molecules participate in a signaling pathway known as the Wnt pathway, except that Wnt signaling normally occurs through a pathway known as the canonical Wnt pathway, but Wnt signaling can also occur though so-called non-canonical Wnt signaling pathways. Canonical Wnt signaling involves a secreted glycoprotein called the Wnt, which binds to a receptor. The receptor is a member of the Frizzled gene family and Frizzled binding to Wnt initiates a signaling cascade that culminates in the accumulation of a protein called beta-catenin in the nucleus. Beta-catenin associates with members of the TCF/LEF gene family to change gene expression in the cell. Noncanonical Wnt signaling occurs through changes in Calcium ion concentrations in the cell that elicit changes in cell behavior (see Rao TP, Kühl M Circ Res. 2010;106(12):1798-806).

Experiments in culture showed that reserve HSCs not only are in contact with pre-osteoblasts, but that the Flamingo protein accumulates at the interface between the HSC and the pre-osteoblast. Furthermore, Sugimura and his colleagues showed that Flamingo regulated the distribution of Frizzled8 in the HSC membrane. Other clues were also very curious. Members of the canonical Wnt signaling pathway in reserve HSCs were quite low, but components of the noncanonical Wnt signaling pathway were expressed at high levels in reserve HSCs.

Sugimura explained the significance of these data this way: “These observations indicated that the osteoblast niche provides a microenvironmentr in which non-canonical Wnt signaling prevails over canonical Wnt signaling under normal conditions. It also suggested that Flamingo and Frizzled8 may play a direct role in maintaining the pool of quiescent HSCs.”

Surgimura and his colleagues engineered mice that lacked the Flamingo and Frizzled8 proteins and they found that these mice had very few reserve HSCs. Additionally, HSC function was greatly reduced (>70%).

Treatment of normal mice with the drug 5-fluorouracil, which destroys dividing HSCs, confirmed the role of non-canonical Wnt signaling in the maintenance of reserve HSCs. Under these conditions, noncanonical Wnt signaling components decreased and canonical Wnt signaling components increased as the reserve HSCs transitioned to frequently-dividing HSCs.

Li concluded: “A better understanding of how the balance shifts between the two will provide the necessary mechanistic insight that allows us to reduce non-canonical Wnt-signaling and dial-up canonical Wnt signaling in order to activate quiescent HSCs during aging. But the knob will have to be turned carefully. If the balance shift too far in favor of canonical Wnt signaling, it may well increase the risk of leukemia.”

Mouse Heart-Specific Stem Cells Potentially Offer Hope For Heart Attack Patients

Biomedical researchers from the University of California, San Francisco (UCSF) have published a stem cell experiment in mice that might provide another way to fix damaged heart muscle in heart attack patients. If these results pan out, they could potentially could increase heart function, minimize scar size, promote the growth of new blood vessels around the heart, and doo all this while avoiding the risk to tissue rejection.

Sounds too good to be true? It was published in PLoS ONE. You can read about it here.

To summarize the experiments, researchers isolated a new heart-specific stem cell from the heart tissue of middle-aged mice. When cultured in a laboratory dish, the cells had the ability to differentiate into heart muscle cells that beat in the culture dish. However, these same cells could also become blood vessels, or smooth muscle, which surrounds blood vessels and regulates the diameter of the vessels. All of these tissues are essential for the heart to work and properly function.

After showing that these cells could be grown in the laboratory in converted into heart-specific cell types, this research group examined the ability of these cells to do the same thing inside a living organism. After expanding the cells in culture, they transplanted them into the hearts of sibling mice that had the same genetic lineage as the mice from which the heart stem cells had been isolated in the first place. This prevented the possibility of the immune system of the recipient mice from attacking and rejecting the implanted cells. The implanted stem cells made blood vessels and also formed smooth muscle. The increased blood flow improved heart function.

Even more exciting, these heart specific stem cells are found in all four chambers of the heart. The “cardiosphere-derived cells” (CDCs) that have been used in other clinical trials are only found in the upper chambers of the heart (atria), and express slightly different cell surface proteins. When grown in culture, these cells grow into spheres of cells that are known as “cardiospheres.”  These new heart-specific stem cells are more widely located in the heart, which means that it is possible to isolate them from patients’ hearts by doing ventricular or atrial biopsies. Biopsies of the right ventricle are among the safest procedures for procuring heart cells from live patients. This procedure is relatively easy to perform and does not adversely affect the patient.

The paper’s first author, Jianqin Ye, PhD, MD, senior scientist at UCSF’s Translational Cardiac Stem Cell Program, said, “These findings are very exciting . . . we showed that we can isolate these cells from the heart of middle-aged animals, even after a heart attack. . . we determined that we can return these cells to the animals to induce repair.”

Senior author Yerem Yeghiazarians, MD, director of UCSF’s Translational Cardiac Stem Cell Program and an associate professor at the UCSF Division of Cardiology, agreed with Ye’s assessment: “The finding extends the current knowledge in the field of native cardiac progenitor cell therapy. Most of the previous research has focused on a different subset of cardiac progenitor cells. These novel cardiac precursor cells appear to have great therapeutic potential.”

Yeghiazarians hopes that those patients who have suffered severe heart failure after a heart attack or have enlargement of the heart (cardiomyopathy) could still be treated with their own heart-specific stem cells to improve their overall health and heart function. Because these cells would come from the patients, there would be no concern of cell rejection after therapy.

These heart-specific stem cells are also known as Sca-1+ stem cells. Sca-1 is a small cell surface protein that is involved in cell signaling. These heart-specific Sca-1+ cells also express a transcription factor called Islet (Isl-1). These cells are known to play an important role in heart development. Most of the previous research on heart stem cells has examined different subset of cells known as “c-kit” cells. Sca-1+ cells, like the c-kit cells, are located within a larger clump of cells called cardiospheres.

To isolate the Sca-1+ cells, Yeghiazarians’ group devised ways to separate the Sca-1-expressing cells that were also expressing high levels of Isl-1. Sca-1 is rather easy to use for isolation, since it is a cell surface protein, but Isa-1 is a nuclear protein and is less useful for isolation purposes.

Yeghiazarians proposed that the co-expression of these two molecules that are also made during heart development suggests a strategy for heart therapy: “Heart disease, including heart attack and heart failure, is the number one killer in advanced countries. It would be a huge advance if we could decrease repeat hospitalizations, improve the quality of life and increase survival.” By giving the heart cells that are extremely similar to those cells that help construct it during development; those same cells could reconstruct the heart when it starts to fail.

More studies are on the board for the future, and these animal studies might lead to future clinical trials.

Co-culturing Immune Cells with Umbilical Cord Stem Cells Reverses Type 1 Diabetes in a Small Study

Type 1 diabetes results from an inability to make sufficient quantities of insulin. Insulin is made by specific cells in the pancreatic islets (also known as the islet of Langerhans). Most type 1 diabetics have suffered destruction of their pancreatic beta cells. Beta cell destruction can result from physical trauma to the pancreas, which causes the digestive enzymes of the pancreas to destroy the beta cells. For example, pancreatitis, pancreatic surgery, or certain industrial chemicals can cause diabetes. Also, particular drugs can also cause temporary diabetes, such as corticosteroids, beta blockers, and phenytoin. Rare genetic disorders (Klinefelter syndrome, Huntington’s chorea, Wolfram syndrome, leprechaunism, Rabson-Mendenhall syndrome, lipoatrophic diabetes, and others) and hormonal disorders (acromegaly, Cushing syndrome, pheochromocytoma, hyperthyroidism, somatostatinoma, aldosteronoma) also increase the risk for diabetes.

Additionally, viral infections of pancreas can cause the immune response to destroy pancreatic cells, and this wipes out enough beta cells to cause the onset of type 1 diabetes. The coxsackievirus family of viruses is a family of enteric viruses that are cause infections that are sometimes associated with the onset of type 1 diabetes, as are mumps and congenital rubella. In most cases, genetic factors cause the immune system to view the pancreatic beta cells as foreign invaders, and the beta cells are attacked and destroyed. Researchers have found at least 18 genetic loci that are designated IDDM1 – IDDM18 that are related to type 1 diabetes. The IDDM1 region contains the “HLA genes” that encode proteins called “major histocompatibility complex”. HLA genes encode cell-surface proteins that act as “bar codes” for the immune system. When cells have the proper bar codes on their cell surfaces, the immune system recognizes those cells as being a part of the body in which they reside, and the immune system leaves them alone. Any cells that do not have the right bar codes are attacked and destroyed, which is known as the “graft versus host response.” Therefore, it is fair to say that HLA genes affect the immune response. New advances in genetic research are identifying other genetic components of type 1 diabetes. Other chromosomes and genes continue to be identified.

A recent paper attempts to cure type 1 diabetes by using umbilical cord stem cells. Umbilical cord stem cells (UCSCs) have the ability to greatly calm down the immune system. UCSCs secrete a wide variety of molecules that prevent immune cells from reacting to and destroying other cells, and also have many cell surface proteins that bind to the surfaces of immune cells and put them to sleep (see Abdi et al., Diabetes 2008;57:1759-67 & Aguayo-Mazzucato C. and Bonner-Weir S, Nature Reviews Endocrinology 2010;6:726-36).

Animal experiments have shown that co-culturing UCSCs with circulating immune cells alters the immune response against pancreatic beta cells and greatly increases the ability of the animal to regulate blood glucose levels (Zhao et al., PLoS ONE 2009;4:e4226). The UCSCs seem to “re-educate” the immune cells so that they do not recognize the pancreatic islets are foreign anymore. Therefore, Yong Zhao and his colleagues in Theodore Mazzone’s laboratory at the University of Chicago, IL, and collaborators at the General Hospital of Jinan Military Command, Shandong, China, used human UCSCs to re-educate immune cells in human type 1 diabetic patients.  See here for this paper.

To do this, they circulated the blood of each patient through a close-loop system that separated the immune cells (lymphocytes) from whole blood. Thee lymphocytes were then co-cultured with UCSCs for 2-3 hours and then returned to the patients.

The results were remarkable. Six patients in group A, who all had some residual beta cell function showed successively improved insulin production 12-24 weeks after treatment. They also showed a reduced need for insulin shots, and overall improvement of their fasting blood glucose levels. Six patients in group B, who had no residual beta cell function, showed increased production of insulin production 12 week after treatment. This is an incredible finding because those without beta cells essentially grew new ones that were not attacked by the immune system. The group B group also saw successively reduced requirements for injected insulin. The patients in the control, whose immune cells did not undergo re-education by UCSCs showed no improvement.

Furthermore, the patients whose immune cells were re-educated by the UCSCs, did not experience any adverse effects. This procedure seems to be quite safe and feasible.

There is a word of caution here. These patients must be followed over several years to establish that the re-education of the lymphocytes is maintained over time. Also, this study is quite small and despite the amazing results, a larger study is needed. All the same, this is an incredible result that reverses type 1 diabetes, and even though caution is needed, embryonic stem cells were not required to do this.